Modified surface antigen

ABSTRACT

Novel proteins that constitute modified forms of a  Neisseria meningitidis  surface antigen and encoding nucleic acids are provided. The modified surface proteins are characterized by having deletions of non-conserved amino acids, and thereby being capable of eliciting cross-protective immune responses against  Neisseria meningitidis . The invention extends to the use of the modified surface antigens in diagnostics, in therapeutic and prophylactic vaccines and in the design and/or screening of medicaments. The modified surface antigens are particularly useful in vaccines which effectively immunize against a broader spectrum of  N. meningitidis  strains than would be expected from a corresponding wild-type surface antigen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the priority ofcopending U.S. patent application Ser. No. 09/771,382, filed Jan. 25,2001, which in turn was a non-provisional application based on andentitled to priority, pursuant to 35 U.S.C. §119(e), of U.S. ProvisionalApplication Ser. No. 60/177,917, filed Jan. 25, 2000, which priority ishereby claimed. The disclosure of these related applications are herebyincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to novel proteins that constitute modified formsof a Neisseria meningitidis surface antigen, to nucleic acids encodingsuch novel peptides and polypeptides, and to the use of these indiagnostics, in therapeutic and prophylactic vaccines and in the designand/or screening of medicaments. More particularly, by having deletionsof non-conserved amino acids, the modified surface antigens of theinvention may be useful in vaccines which effectively immunize against abroader spectrum of N. meningitidis strains than would be expected froma corresponding wild-type surface antigen.

BACKGROUND OF THE INVENTION

Neisseria meningitidis is a Gram-negative bacterium and the causativeagent of meningococcal meningitis and septicemia. Its only known host isthe human, and it may be carried asymptomatically by approximately 10%of the population (Caugant et al, 1994, Journal of Clinical Microbiology32 323).

N. meningitidis may express a polysaccharide capsule, and this allowsclassification of the bacteria according to the nature of the capsuleexpressed. There are at least twelve serogroups of N. meningitidis: A,B, C, 29-E, H, I, K, L, W135, X, Y and Z, of which serogroups A, B, andC cause 90% of meningococcal disease (Poolman et al, 1995, InfectiousAgents and Disease 4 13). Vaccines directed against serogroups A and Care available, but the serogroup B capsular polysaccharide is poorlyimmunogenic and does not induce protection in humans. Other membrane andextracellular components are therefore being examined for theirsuitability for inclusion in vaccines. Examples include the outermembrane proteins of classes 1, 2 and 3 (porin; encoded by por genes),and classes 4 (Rmp) and 5 (Opacity proteins; encoded by opa and opcgenes). However, to date, none of these candidates is able to inducecomplete protection, particularly in children (Romero et al., 1994,Clinical Microbiology Review, 7 559; Poolman et al., 1995, supra).

To create an effective vaccine, it is necessary to identify componentsof N. meningitidis which are present in a majority of strains, and whichare capable of inducing a protective immune response (for example,bactericidal antibodies).

In this regard, reference is made to International Publications WO99/24578, WO99/36544, WO99/58683 and WO99/57280, each of which isincorporated herein by reference and describe a number of candidateproteins that may be useful in vaccines to immunize against Neisseriameningitidis.

In this regard, particular reference is made to InternationalPublication WO99/31132 and Peak et al. 2000, FEMS Immunol. Med.Microbiol. 28 329, each of which is incorporated herein by reference anddescribe a novel surface antigen isolated from a number of differentstrains of N. meningitidis, which surface antigen, and allelic variantsthereof, for the purposes of this specification will be referred to asNhhA.

SUMMARY OF THE INVENTION

The present inventors have discovered that the NhhA surface antigen haspolypeptide regions which are variable between N. meningitidis strains,and other regions which are conserved between strains. The variableregions may be immunogenic and tend to elicit strain-specific immuneresponses, such that vaccines incorporating an NhhA antigen derived froma particular strain of N. meningitidis tend to preferentially immunizeagainst that particular strain. As a result, the present inventors havesought to produce a modified NhhA polypeptide which elicits an immuneresponse which is not as strain-specific as that elicited by wild-typeNhhA. This modified NhhA antigen will be useful for the production oftherapeutic and/or prophylactic vaccines against N. meningitidis as willbe described hereinafter. By directing the immune response primarilyagainst conserved epitopes, such vaccines should effectively immunizeagainst a broader spectrum of N. meningitidis strains than would beexpected following immunization with wild-type NhhA.

The present invention is therefore broadly directed to isolated proteinshaving conserved amino acids of NhhA polypeptides.

Proteins of the invention may therefore have one or more deletions ofnon-conserved amino acids compared to a corresponding wild-type NhhApolypeptide.

In a first aspect, the invention provides an isolated protein comprisingtwelve or more contiguous conserved amino acids sequences of an NhhApolypeptide, said isolated protein excluding wild-type NhhApolypeptides.

Suitably, the protein of the invention is capable of eliciting an immuneresponse.

Preferably, the immune response is less strain-specific than thatelicited by said corresponding wild-type NhhA polypeptide.

More preferably, said immune response provides protection against one ormore strains of N. meningitidis, or even more preferably a plurality ofstrains of N. meningitidis.

Wild-type NhhA polypeptide sequences are exemplified in FIG. 1 (SEQ IDNOS:1 to 10).

A consensus amino acid sequence is also set forth in FIG. 1 (SEQ IDNO:1).

The isolated protein of the invention preferably comprises one or moreconstant regions of an NhhA polypeptide, herein designated C1, C2, C3,C4 and C5 regions in FIG. 1.

It will be appreciated that according to this aspect, suitably one ormore non-conserved amino acids of a variable region of an NhhApolypeptide, designated as V1, V2, V3 or V4 regions in FIG. 1, aredeleted with respect to a wild-type NhhA polypeptide.

Preferably, a V1 region, or at least a substantial portion thereof, isdeleted.

In particular embodiments, the isolated protein has an amino acidsequence as set forth in any one of FIGS. 5 to 9 (SEQ ID NOS:23 to 27)which are examples of “modified NhhA polypeptides of the invention”. InFIGS. 14A-14G (SEQ ID NOS:33 to 39) further examples are provided of“mature” polypeptides predicted to result of removal of N-terminalsignal sequences.

According to a second aspect, the invention provides an isolated nucleicacid encoding a polypeptide according to the first aspect.

Wild-type nhhA nucleic acid sequences are exemplified in FIG. 2 (SEQ IDNOS:12 to 21).

A consensus nucleic acid sequence is also set forth in FIG. 2 (SEQ IDNO:22).

Preferably, the C1, C2, C3, C4 and C5 regions are encoded by respectivenucleotide sequences as set forth in FIG. 2.

Preferably, the V1, V2, V3 and V4 regions are encoded by respectivenucleotide sequences as set forth in FIG. 2.

In a particular embodiment, the isolated nucleic acid of the inventionhas a nucleotide sequence as set forth in any one of FIGS. 5 to 9 (SEQID NOS:28 to 32), which are particular examples of “modified nhhAnucleic acids of the invention”.

The invention according to the first and second aspects extends tohomologs, fragments, variants and derivatives of the isolated proteinsand nucleic acids of the invention.

Specifically excluded from the scope of the invention are wild-type NhhApolypeptides and nhhA nucleic acids.

In a third aspect, the invention resides in an expression constructcomprising an expression vector and a nucleic acid according to thesecond aspect, wherein said sequence is operably linked to one or moreregulatory nucleic acids in said expression vector.

In a fourth aspect, the invention provides a host cell containing anexpression construct according to the third aspect.

In a fifth aspect of the invention, there is provided a method ofproducing a recombinant isolated protein according to the first aspect,said method comprising the steps of:

(i) culturing a host cell containing an expression vector according tothe third aspect such that said polypeptide is expressed in said hostcell; and

(ii) isolating said recombinant polypeptide.

In a sixth aspect, the invention provides an antibody or antibodyfragment that binds to a protein of the invention, fragment, variant orderivative thereof.

In a seventh aspect, the invention provides a method of detecting N.meningitidis in a biological sample suspected of containing same, saidmethod comprising the steps of:—

(i) isolating the biological sample from an individual;

(ii) combining the above-mentioned antibody or antibody fragment withthe biological sample; and

(iii) detecting specifically bound antibody or antibody fragment whichindicates the presence of N. meningitidis.

In an eighth aspect, there is provided a method of detecting N.meningitidis bacteria in a biological sample suspected of containingsaid bacteria, said method comprising the steps of:—

-   -   (i) isolating the biological sample from a patient;    -   (ii) detecting a nucleic acid sequence according to the        second-mentioned aspect in said sample which indicates the        presence of said bacteria.

In a ninth aspect, the invention provides a method for diagnosinginfection of an individual by N. meningitidis, said method comprisingthe steps of:—

(i) contacting a biological sample from an individual with apolypeptide, fragment, variant or derivative of the invention; and

(ii) determining the presence or absence of a complex between saidpolypeptide, fragment, variant or derivative and N.meningitidis-specific antibodies in said sample, wherein the presence ofsaid complex is indicative of said infection.

Preferably, the individual is a mammal.

More preferably, the individual is a human.

In a tenth aspect, the invention also extends to the use of an isolatedprotein according to the first-mentioned aspect, the use of isolatednucleic acids according to the second aspect or the use of the antibodyor antibody fragment mentioned above in a kit for detecting N.meningitidis bacteria in a biological sample.

In an eleventh aspect of the invention, there is provided apharmaceutical composition comprising an isolated protein according tothe first mentioned aspect.

Preferably, said pharmaceutical composition is a vaccine.

In a twelfth aspect, the invention provides a method of preventinginfection of a patient by N. meningitidis, comprising the step ofadministrating a pharmaceutically effective amount of theabove-mentioned vaccine.

In a thirteenth aspect, the invention provides a method of identifyingan immunogenic fragment of an isolated protein, variant or derivativeaccording to the first mentioned aspect, comprising the steps of:—

(i) producing a fragment of said polypeptide, variant or derivative;

(ii) administering said fragment to an individual; and

(iii) detecting an immune response in said individual, which responseincludes production of elements which specifically bind N. meningitidisand/or said polypeptide, variant or derivative, and/or a protectiveeffect against N. meningitidis infection.

Preferably, the individual is a mammal.

More preferably, the individual is a human.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

Table 1: Identification of amino acids of the conserved regions (C1, C2,C3, C4 and C5) and variable regions (V1, V2, V3 and V4) of an NhhApolypeptide from each often (10) indicated strains of N. meningitidis.Relevant SEQ ID NOS are also indicated. Column 1=strain designation. SEQID NOS:1-9 were previously described in copending applicationWO99/31132; the sequences of NhhA and nhhA of strain Z2491 were obtainedfrom the database of the Wellcome Trust/Sanger Institute genomicsequencing project for N. meningitidis; column 2=amino acid numbering ofC1 region; column 3=amino acid numbering of V1 region; column 4=aminoacid numbering of C2 region; column 5=amino acid numbering of V2 region;column 6=amino acid numbering of C3 region, column 7=amino acidnumbering of V2 region; column 8=amino acid numbering of C4 region;column 9=amino acid numbering of V4 region; column 10=amino acidnumbering of C5 region. Note that the amino acid numbering of theconsensus sequence (SEQ ID NO:11) is also indicated.

Table 2: Table of amino acid substitutions.

FIG. 1 (comprising FIGS. 1A-1E): Amino acid sequence alignments of NhhApolypeptide amino acid sequences from ten (10) N. meningitidis strains(SEQ ID NOS:1-10) together with consensus sequence (SEQ ID NO:11).Strain names and polypeptide sequences used in this alignment correspondto the strain names and SEQ ID NOS in column 1 of Table 1. Amino acidsare indicated by standard single letter abbreviations. Consensus aminoacids are shown only where residues are completely conserved. Conservedregions (double underlined, labeled C1, C2, C3, C4, C5) and variableregions (single underlined, labeled V1, V2, V3, V4) are indicated underthe consensus sequence.

FIG. 2 (comprising FIGS. 2A-2H): Nucleotide sequence alignment of nhhAnucleic acids from ten (10) N. meningitidis strains, which sequencesencode the amino acid sequences of FIG. 1. Regions C1, C2, C3, C4, C5and V1, V2, V3, V4 are as described in FIG. 1 and Table 1.

FIG. 3 (comprising FIGS. 3A and 3B): Plasmid map corresponding to pCO14Kwith a PCR amplification product encoding wild-type PMC21 NhhA operablylinked to the porA promoter. (Not drawn to scale) FIG. 3A: Solid arrowsindicate the arrangement of the porA and kanR genes inpCO14K.Oligonucleotide primers HOMP5′ and HOMP3′AN used to amplify thenhhA gene of strain PMC21 are shown. The nhhA gene is shown by dottedarrow, the porA promoter by a black box, and Eagl and NcoI restrictionsites used to replace porA with nhhA in as described in Example 2 areshown. FIG. 3B Arrangement of genes in pIP52(PMC21), as described inExample 2. The BglII site used to construct a mutant as described inExample 4 is shown.

FIG. 4 (comprising FIGS. 4A-4C): Schematic representation of SpliceOverlap Extension PCR strategy for deletion of specific regions of NhhApolypeptides. A schematic of the wild-type nhhA gene is shown at the topof FIGS. 4A-C, and the recombinant nhhA is shown at the bottom of thesefigures, with variable regions shown as black and constant regions byunfilled boxes. Arrows indicate approximate location of oligonucleotideprimers. Vertical hatched lines indicate amplification products. Whereoligonucleotide sequence is from discontinuous regions of an nhhAnucleic acid, this is shown by a dotted line between such discontinuousregions. Approximate scale indicated. Double vertical lines indicatethat only a portion of the C5 region is shown. FIG. 4A: shows thestrategy as described in Example 6. FIG. 4B: shows the strategy asdescribed in Example 7. FIG. 4C: shows the strategy as described inExample 8.

FIG. 5 (comprising FIGS. 5A and 5B): FIG. 5A Amino acid sequence of PMC21 NhhA deletion mutant polypeptide (SEQ ID NO:23) produced in Example4; and FIG. 5B encoding nucleotide sequence (SEQ ID NO:28).

FIG. 6 (comprising FIGS. 6A and 6B): FIG. 6A Amino acid sequence of H41NNhhA deletion mutant polypeptide (SEQ ID NO:24) produced in Example 5;and FIG. 6B encoding nucleotide sequence (SEQ ID NO:29).

FIG. 7 (comprising FIGS. 7A and 7B): FIG. 7A Amino acid sequence ofPMC21 NhhA deletion mutant polypeptide (SEQ ID NO:25) produced by spliceoverlap PCR in Example 6; and FIG. 7B encoding nucleotide sequence (SEQID NO:30).

FIG. 8 (comprising FIGS. 8A and 8B): FIG. 8A Amino acid sequence ofPMC21 NhhA deletion mutant polypeptide (SEQ ID NO:26) produced by spliceoverlap PCR in Example 7; and FIG. 8B encoding nucleotide sequence (SEQID NO:31).

FIG. 9 (comprising FIGS. 9A and 9B): FIG. 9A Amino acid sequence ofPMC21 NhhA deletion mutant polypeptide (SEQ ID NO: 27) produced bysplice overlap PCR in Example 8; and FIG. 9B encoding nucleotidesequence (SEQ ID NO:32).

FIG. 10 (comprising FIGS. 10A and 10B): Amino acid sequence alignmentsof wild type and NhhA deletion mutant polypeptide sequences. Thesepolypeptides were produced as described in Example 2, Example 3, Example4 and Example 5. Amino acids are indicated by the one letterabbreviation. Conserved regions labeled C1, C2, C3, C4 and C5corresponding to those defined in Table 1 and FIG. 1 are indicated bydouble underlining of full length sequences from H41 and PMC21, andvariable regions labeled V1, V2, V3, V4 corresponding to those definedin Table 1 and FIG. 1 are indicated by single underlining of full lengthsequences from H41 and PMC21.

FIG. 11: Western immunoblot showing over expressed NhhA. 45 μg totalcell protein was separated on 4-20% gradient SDS-PAGE before transfer toa nitrocellulose filter and western immunoblot as described in Example9. Lane 1: Parental strain showing wild-type level of NhhA expression.Lane 2: Strain P6 (overexpresses PMC 21 NhhA as described in Example 2).Lane 3: Strain PΔ6 (overexpresses the truncated PMC 21NhhA described inExample 4). Lane 4: Strain H14 (overexpresses H41 NhhA described inExample 3). Lane 5: Strain HΔ8 (overexpresses the truncated H41 NhhAdescribed in Example 5). Lane 6: Strain 2A (NhhA expression abolished bymutation of nhhA gene as described in International PublicationWO99/31132). Migration of standards is indicated: 185 kDa, 119 kDa, 85kDa, 62 kDa, 51.2 kDa, 38.2 kDa, 22.4 kDa. Wild-type NhhA polypeptide ispresent as a high molecular weight immunoreactive band present in lane 1but absent from lane 6.

FIG. 12: Isolated NhhA deletion mutant polypeptides. NhhA polypeptideswere isolated as described in Example 9 before separation on 4-20%SD-PAGE. The polyacrylamide gel was Coomassie stained. Lane 1: OMCpreparation of Strain overexpressing the truncated PMC21 NhhApolypeptide described in Example 6. Lane 2: Purified truncated PMC21NhhA polypeptide. Lane 3: OMC preparation of Strain over-expressing thetruncated PMC21 NhhA polypeptide described in Example 4. Lane 4:Purified truncated PMC21 NhhA polypeptide. Lane 5: OMC preparation of astrain overexpressing PMC21 NhhA polypeptide described in Example 2.Lane 6: Purified PMC21 NhhA polypeptide. Lane 7: Molecular weightstandards of 173 kDa, 111 kDa, 80 kDa, 61 kDa, 49 kDa, 36 kDa. Note thatthe reactive high molecular weight species in all lanes except 6probably represents multimers of NhhA polypeptides. Other bands areprobably less stable forms of NhhA or breakdown products. Note these areabsent from lane 6.

FIG. 13 (comprising FIGS. 13A-13C): Western Immunoblot using anti-NhhAprotein mouse sera. In all of FIGS. 13A-13C, lanes 1, 3, 5, 7, containOMC of Strain over expressing PMC21 NhhA polypeptide, and lanes 2, 4, 6,and 8 contain OMC of strain 2A which does not express NhhA. FIG. 13A:Lanes 1 and 2: mouse A inoculated with wild-type PMC21 NhhA at a 1:1000dilution. Lanes 3 and 4: mouse A inoculated with wild-type PMC21 NhhA ata 1:10.000 dilution. Lanes 5 and 6, mouse B inoculated with wild-typePMC21 NhhA at a 1:1000 dilution. Lanes 7 and 8: mouse B inoculated withwild-type PMC21 NhhA at a 1:10.000 dilution. FIG. 13B: Lanes 1 & 2:mouse C inoculated with truncated PMC21 NhhA polypeptide (Example 4) ata 1:1000 dilution. Lanes 3 & 4: mouse C inoculated with truncated PMC21NhhA polypeptide (Example 4) at a 1:10,000 dilution. Lanes 5 & 6: mouseD inoculated with truncated PMC21 NhhA (Example 4) at a 1:1000 dilution.Lanes 7 and 8: mouse D inoculated with truncated PMC21 NhhA (Example 4)at a 1:1000 dilution. FIG. 13C: Lanes 1 & 2: mouse E inoculated withtruncated PMC21 NhhA (Example 6) at a 1:1000 dilution. Lanes 3 and 4:mouse E inoculated with truncated PMC21 NhhA (Example 6) at a 1:10,000dilution. Lanes 5 & 6: mouse F inoculated with truncated PMC21 NhhA(Example 6) at a 1:1000 dilution. Lanes 7 & 8: mouse F inoculated withtruncated PMC21 NhhA (Example 6) at a 1:1000 dilution.

FIG. 14 (comprising FIGS. 14A-14G): Predicted mature NhhA polypeptidedeletion mutants. FIG. 14A: predicted mature protein described inExample 2 (SEQ ID NO:33); FIG. 14B: predicted mature protein describedin Example 3 (SEQ ID NO:34); FIG. 14C: predicted mature proteindescribed in Example 4 (SEQ ID NO:35); FIG. 14D: predicted matureprotein described in Example 5 (SEQ ID NO:36); FIG. 14E: predictedmature protein described in Example 6 (SEQ ID NO:37); FIG. 14F:predicted mature protein described in Example 7 (SEQ ID NO:38); and FIG.14G: predicted mature protein described in Example 8 (SEQ ID NO:39).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

With regard to nomenclature, NhhA is used herein when reference is madeto proteins of the invention, while nhhA is used herein when referenceis made to nucleic acids of the invention. It will also be understoodthat NhhA/nhhA proteins and nucleic acids include the HiaNm/hianmproteins and nucleic acids referred to in WO99/31132, for example,without limitation thereto.

The present invention is predicated, at least in part, by theelucidation of conserved and less-conserved regions in the NhhApolypeptide in ten (10) strains of N. meningitidis. Correspondingregions are predicted to be conserved in other allelic variants of theexemplified NhhA polypeptides.

It will be appreciated that central to the present invention is therealization that by deleting non-conserved amino acids in a wild-typeNhhA polypeptide to form a modified NhhA polypeptide of the invention,an immune response may be elicited upon immunization by said polypeptideof the invention which, by directing the immune response againstconserved epitopes, will provide protection against one or moreheterologous strains of N. meningitidis.

As used herein, “non-conserved” amino acids are amino acid residuespresent in a wild-type NhhA polypeptide from a first N. meningitidisstrain, but which are not present in a wild-type NhhA polypeptide fromone or more other strains.

Suitably, the polypeptides of the first aspect have at least a portionof one of the V1, V2, V3 or V4 regions deleted with respect to thecorresponding wild-type sequence, and accordingly, may be collectivelyreferred to as examples of “deletion mutants”.

It will be appreciated that the present inventors have identified theV1, V2, V3 and V4 regions as being regions of wild-type NhhApolypeptides having relatively high frequencies of non-conserved aminoacids compared to the relatively conserved C1-5 regions.

Of the V regions, the V1 (hypervariable) and V2 regions have the highestfrequency of non-conserved amino acids, while V3 and V4 have relativelylower frequencies. However, the V1 region constitutes a more significantproportion of wild-type NhhA polypeptides than does the V2 region (interms of total amino acids). Therefore, it is preferred that theisolated proteins according to the first-mentioned aspect have at leasta substantial portion of the V1 region deleted.

It will also be realized by the skilled person that in constructing saiddeletion mutants, “shuffling” of regions between NhhA polypeptides ofdifferent N. meningitidis strains is possible. For example, an NhhApolypeptide of the invention may comprise a H41 C1 region together witha PMC21 C5 region.

Such “shuffling” is particularly well-suited to recombinant DNA methods.

For the purposes of this invention, by “isolated” is meant material thathas been removed from its natural state or otherwise been subjected tohuman manipulation. Isolated material may be substantially oressentially free from components that normally accompany it in itsnatural state, or may be manipulated so as to be in an artificial statetogether with components that normally accompany it in its naturalstate. Isolated material may be in native or recombinant form.

By “protein” is meant an amino acid polymer. The amino acids may benatural or non-natural amino acids as are well understood in the art.

A “peptide” is a protein having no more than fifty (50) amino acids.

A polypeptide is a protein having fifty (50) or more amino acids.

As used herein, the phrase “elicits an immune response” refers to theability of an isolated polypeptide of the invention to produce an immuneresponse in a mammal to which it is administered, wherein the responseis directed to N. meningitidis and/or said polypeptide. Preferably, theimmune response includes production of bactericidal antibodies. Morepreferably, the immune response is protective against N. meningitidisinfection.

“Strain-specific” is used herein in the context of an immune responsewhich is directed to, or at least predominantly directed to, anautologous N. meningitidis strain.

As used herein, “cross-reactive” means an ability of a polypeptide ofthe invention to elicit an immune response directed to one or moreheterologous N. meningitidis strains.

As used herein, “cross-protective” means an ability of a polypeptide ofthe invention to elicit an immune response and thereby provideprotection against infection by one or more heterologous N. meningitidisstrains.

Therefore, in light of the foregoing, said polypeptide of the inventionmay be referred to herein as an “immunogen”, or as being “immunogenic”.

Although for the purposes of the present invention, said modified NhhAproteins have been exemplified by the amino acid sequences set forth inFIGS. 5 to 9 (SEQ ID NOS:23-27) and FIG. 14, the present invention alsocontemplates fragments, derivatives and variants (such as allelicvariants) of the exemplified proteins.

For example, amino acids can be deleted from any of the C1-5 sequencesset forth in FIG. 1, while not all non-conserved amino acids in the V1-4regions need be deleted in order to reduce strain-specificimmunogenicity. Therefore, isolated proteins of the invention mayinclude fragments of the C1-5 and V1-4 regions.

Indeed, as will be described hereinafter in the Examples, it may beadvantageous for the purposes of recombinant DNA-based production ofpolypeptides of the invention, to delete one or a few amino acids of aC1, C2, C3, C4 and/or C5 region or a V1, V2, V3 and/or V4 region in theinterests of utilizing convenient restriction endonuclease sites andachieving high level expression of stable, immunogenic protein.

In one embodiment, a “fragment” includes an amino acid sequence thatconstitutes less than 100%, but at least 20%, preferably at least 50%,more preferably at least 80% or even more preferably at least 90% ofsaid C1, C2, C3, C4 or C5 regions.

Fragments, for example, may be peptides comprising as few as twelveamino acids such as the C2 region (SEQ ID NO:11) or sequences of atleast twenty contiguous amino acids, or more than one hundred contiguousamino acids corresponding to some or all of the C1, C2, C3, C4 and/or C5regions described herein.

Other fragments exemplified herein are modified NhhA polypeptides of theinvention which have undergone post-translational processing to form amature polypeptide, such as shown in FIG. 14.

In another embodiment, a “fragment” is a small peptide, for example ofat least 6, preferably at least 10 and more preferably at least 20 aminoacids in length, which comprises one or more antigenic determinants orepitopes derived from modified NhhA proteins of the invention. Largerfragments comprising more than one peptide are also contemplated, andmay be obtained through the application of standard recombinant nucleicacid techniques or synthesized using conventional liquid or solid phasesynthesis techniques. For example, reference may be made to solutionsynthesis or solid phase synthesis as described, for example, in Chapter9 entitled “Peptide Synthesis” by Atherton and Shephard which isincluded in a publication entitled “Synthetic Vaccines” edited byNicholson and published by Blackwell Scientific Publications.Alternatively, peptides can be produced by digestion of a polypeptide ofthe invention with proteinases such as endoLys-C, endoArg-C, endoGlu-Cand staphylococcins V8-protease. The digested fragments can be purifiedby, for example, high performance liquid chromatographic (HPLC)techniques.

As used herein, “variant” polypeptides are polypeptides of the inventionin which one or more amino acids have been replaced by different aminoacids. It is well understood in the art that some amino acids may bechanged to others with broadly similar properties without changing thenature of the activity of the polypeptide (conservative substitutions).Exemplary conservative substitutions in the polypeptide may be madeaccording to Table 2.

Substantial changes in function are made by selecting substitutions thatare less conservative than those shown in Table 2. Other replacementswould be non-conservative substitutions and relatively fewer of thesemay be tolerated. Generally, the substitutions which are likely toproduce the greatest changes in a polypeptide's properties are those inwhich (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for,or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val); (b) acysteine or proline is substituted for, or by, any other residue; (c) aresidue having an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp) or(d) a residue having a bulky side chain (e.g., Phe or Trp) issubstituted for, or by, one having a smaller side chain (e.g., Ala, Ser)or no side chain (e.g., Gly).

The term “variant” also includes NhhA polypeptides of the inventionproduced from allelic variants of the sequences exemplified in thisspecification.

NhhA polypeptide variants may fall within the scope of the term“polypeptide homologs”.

Polypeptide homologs share at least 70%, preferably at least 80% andmore preferably at least 90% sequence identity with the amino acidsequences of modified NhhA polypeptides of the invention as hereinbeforedescribed.

As generally used herein, a “homolog” shares a definable nucleotide oramino acid sequence relationship with a nucleic acid or polypeptide ofthe invention as the case may be.

For example, such homologs are contemplated as having amino acidsequences that differ from those exemplified herein, but which areimmunogenic and provide cross-protective immunity.

Specifically excluded from the scope of the term “homologs” arewild-type NhhA polypeptides and nhhA nucleic acids.

Included within the scope of homologs are “orthologs”, which arefunctionally-related polypeptides and their encoding nucleic acids,isolated from bacterial species other than N. meningitidis.

Terms used herein to describe sequence relationships between respectivenucleic acids and polypeptides include “comparison window”, “sequenceidentity”, “percentage of sequence identity” and “substantial identity”.Because respective nucleic acids/polypeptides may each comprise (1) onlyone or more portions of a complete nucleic acid/polypeptide sequencethat are shared by the nucleic acids/polypeptides, and (2) one or moreportions which are divergent between the nucleic acids/polypeptides,sequence comparisons are typically performed by comparing sequences overa “comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment oftypically 12 contiguous residues that is compared to a referencesequence. The comparison window may comprise additions or deletions(i.e., gaps) of about 20% or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the respective sequences. Optimal alignment of sequences for aligninga comparison window may be conducted by computerised implementations ofalgorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, 575 Science Drive Madison, Wis., USA,incorporated herein by reference) or by inspection and the bestalignment (i.e., resulting in the highest percentage homology over thecomparison window) generated by any of the various methods selected.Reference also may be made to the BLAST family of programs as forexample disclosed by Altschul et al., 1997, Nucl. Acids Res. 253389,which is incorporated herein by reference.

A detailed discussion of sequence analysis can be found in Unit 19.3 ofCURRENT PROTOCOLS 1N MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley &Sons Inc NY, 1995-1999).

The term “sequence identity” is used herein in its broadest sense toinclude the number of exact nucleotide or amino acid matches havingregard to an appropriate alignment using a standard algorithm, havingregard to the extent that sequences are identical over a window ofcomparison. Thus, a “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, I) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For example, “sequence identity” may be understood tomean the “match percentage” calculated by the DNASIS computer program(Version 2.5 for windows; available from Hitachi Software engineeringCo., Ltd., South San Francisco, Calif., USA).

Thus, it is well within the capabilities of the skilled person toprepare polypeptide homologs of the invention, such as variants ashereinbefore defined, by recombinant DNA technology. For example,nucleic acids of the invention can be mutated using either randommutagenesis for example using transposon mutagenesis, or site-directedmutagenesis. The resultant DNA fragments are then cloned into suitableexpression hosts such as E. coli using conventional technology andclones that retain the desired activity are detected. Where the cloneshave been derived using random mutagenesis techniques, positive cloneswould have to be sequenced in order to detect the mutation.

As used herein, “derivative” polypeptides are polypeptides of theinvention which have been altered, for example by conjugation orcomplexing with other chemical moieties or by post-translationalmodification techniques as would be understood in the art. Suchderivatives include amino acid deletions and/or additions to NhhApolypeptides of the invention, or variants thereof, wherein saidderivatives elicit an immune response.

“Additions” of amino acids may include fusion of the polypeptides orvariants thereof with other polypeptides or proteins. In this regard, itwill be appreciated that the polypeptides or variants of the inventionmay be incorporated into larger polypeptides, and such largerpolypeptides may also be expected to be immunogenic. The polypeptides asdescribed above may be fused to a further protein, for example, which isnot derived from N. meningitidis. The other protein may, by way ofexample, assist in the purification of the protein. For instance apolyhistidine tag, or a maltose binding protein may be used.Alternatively, it may produce an immune response which is effectiveagainst N. meningitidis or it may produce an immune response againstanother pathogen. Other possible fusion proteins are those which producean immunomodulatory response. Particular examples of such proteinsinclude Protein A or glutathione S-transferase (GST). In addition, thepolypeptide may be fused to an oligosaccharide based vaccine componentwhere it acts as a carrier protein.

Other derivatives contemplated by the invention include, but are notlimited to, modification to side chains, incorporation of unnaturalamino acids and/or their derivatives during peptide, polypeptide orprotein synthesis and the use of crosslinkers and other methods whichimpose conformational constraints on the polypeptides, fragments andvariants of the invention. Examples of side chain modificationscontemplated by the present invention include modifications of aminogroups such as by acylation with acetic anhydride; acylation of aminogroups with succinic anhydride and tetrahydrophthalic anhydride;amidination with methylacetimidate; carbamoylation of amino groups withcyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed byreduction with NaBH₄; reductive alkylation by reaction with an aldehydefollowed by reduction with NaBH₄; and trinitrobenzylation of aminogroups with 2,4,6-trinitrobenzene sulphonic acid (TNBS).

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitization, by way ofexample, to a corresponding amide.

The guanidine group of arginine residues may be modified by formation ofheterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

Sulphydryl groups may be modified by methods such as performic acidoxidation to cysteic acid; formation of mercurial derivatives using4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate;2-chloromercuri-4-nitrophenol, phenylmercury chloride, and othermercurials; formation of a mixed disulphides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; carboxymethylation with iodoacetic acid or iodoacetamide; andcarbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified, for example, by alkylation of theindole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides orby oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified byN-carbethoxylation with diethylpyrocarbonate or by alkylation withiodoacetic acid derivatives.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include but are not limited to, use of 4-amino butyricacid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine,norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/orD-isomers of amino acids.

The invention also contemplates covalently modifying a polypeptide,fragment or variant of the invention with dinitrophenol, in order torender it immunogenic in humans.

Isolated proteins of the invention (inclusive of fragments, variants,derivatives and homologs) may be prepared by any suitable procedureknown to those of skill in the art.

For example, the protein may be prepared as a recombinant polypeptide bya procedure including the steps of:

-   -   (i) preparing an expression construct which comprises a modified        nhhA nucleic acid of the invention, operably linked to one or        more regulatory nucleotide sequences;    -   (ii) transfecting or transforming a suitable host cell with the        expression construct; and    -   (iii) expressing the recombinant polypeptide in said host cell.

A number of Examples will be provided hereinafter which describeproduction of modified nhhA nucleic acids of the invention by PCR.

In one particular embodiment, PCR is splice overlap PCR, as will bedescribed hereinafter, which method is based on that described in Ho etal., 1989, Gene 77 51, and by Horton et al., 1989, Gene 77 61, which areboth incorporated herein by reference.

For the purposes of host cell expression, the recombinant nucleic acidis operably linked to one or more regulatory sequences in an expressionvector.

An “expression vector” may be either a self-replicatingextra-chromosomal vector such as a plasmid, or a vector that integratesinto a host genome.

By “operably linked” is meant that said regulatory nucleotidesequence(s) is/are positioned relative to the recombinant nucleic acidof the invention to initiate, regulate or otherwise controltranscription.

Regulatory nucleotide sequences will generally be appropriate for thehost cell used for expression. Numerous types of appropriate expressionvectors and suitable regulatory sequences are known in the art for avariety of host cells.

Typically, said one or more regulatory nucleotide sequences may include,but are not limited to, promoter sequences, leader or signal sequences,ribosomal binding sites, transcriptional start and terminationsequences, translational start and termination sequences, and enhanceror activator sequences.

Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter.

In a preferred embodiment, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectablemarker genes are well known in the art and will vary with the host cellused.

In an embodiment, the expression vector is pCO14K, which has a porApromoter and kanamycin selection gene, as will be described in detailhereinafter. According to this embodiment, the host cell is a bacteriumselected from the group consisting of E. coli and N. meningitidis.

The expression vector may also include a fusion partner (typicallyprovided by the expression vector) so that the recombinant polypeptideof the invention is expressed as a fusion polypeptide with said fusionpartner. The main advantage of fusion partners is that they assistidentification and/or purification of said fusion polypeptide.

In order to express said fusion polypeptide, it is necessary to ligate anucleotide sequence according to the invention into the expressionvector so that the translational reading frames of the fusion partnerand the nucleotide sequence of the invention coincide.

Well known examples of fusion partners include, but are not limited to,glutathione-S-transferase (GST), Fc portion of human IgG, maltosebinding protein (MBP) and hexahistidine (HIS₆), which are particularlyuseful for isolation of the fusion polypeptide by affinitychromatography. For the purposes of fusion polypeptide purification byaffinity chromatography, relevant matrices for affinity chromatographyare glutathione-, amylose-, and nickel- or cobalt-conjugated resinsrespectively. Many such matrices are available in “kit” form, such asthe QIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners andthe Pharmacia GST purification system.

A preferred fusion partner is MBP, which is described hereinafter inExample 11.

Another fusion partner well known in the art is green fluorescentprotein (GFP). This fusion partner serves as a fluorescent “tag” whichallows the fusion polypeptide of the invention to be identified byfluorescence microscopy or by flow cytometry. The GFP tag is useful whenassessing subcellular localization of the fusion polypeptide of theinvention, or for isolating cells which express the fusion polypeptideof the invention. Flow cytometric methods such as fluorescence activatedcell sorting (FACS) are particularly useful in this latter application.

Preferably, the fusion partners also have protease cleavage sites, suchas for Factor X_(a) or Thrombin, which allow the relevant protease topartially digest the fusion polypeptide of the invention and therebyliberate the recombinant polypeptide of the invention therefrom. Theliberated polypeptide can then be isolated from the fusion partner bysubsequent chromatographic separation.

Fusion partners according to the invention also include within theirscope “epitope tags”, which are usually short peptide sequences forwhich a specific antibody is available. Well known examples of epitopetags for which specific monoclonal antibodies are readily availableinclude c-myc, influenza virus haemagglutinin and FLAG tags.

As hereinbefore, polypeptides of the invention may be produced byculturing a host cell transformed with said expression constructcomprising a nucleic acid encoding a polypeptide, or polypeptidehomolog, of the invention. The conditions appropriate for proteinexpression will vary with the choice of expression vector and the hostcell. This is easily ascertained by one skilled in the art throughroutine experimentation.

Suitable host cells for expression may be prokaryotic or eukaryotic. Onepreferred host cell for expression of a polypeptide according to theinvention is a bacterium. The bacterium used may be Escherichia coli orN. meningitidis.

In a preferred embodiment, the host cell is N. meningitidis which hasbeen modified so as to not express PorA, Opa, Opc or capsularpolysaccharide and expresses a desired lipopolysaccharide phenotype.

Alternatively, the host cell may be an insect cell such as, for example,SF9 cells that may be utilized with a baculovirus expression system.

The recombinant protein may be conveniently prepared by a person skilledin the art using standard protocols as for example described inSambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold SpringHarbor Press, 1989), incorporated herein by reference, in particularSections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubelet al., (John Wiley & Sons, Inc. 1995-1999), incorporated herein byreference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS INPROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995-1999)which is incorporated by reference herein, in particular Chapters 1, 5and 6.

Preferred methods of expression of recombinant modified NhhA proteins ofthe invention, and methods for detection of expressed protein, areprovided hereinafter in the Examples.

Nucleotide Sequences

The invention provides an isolated nucleic acid that encodes a modifiedNhhA protein of the invention.

Preferably, said isolated nucleic acid has a nucleotide sequence thatencodes one or more NhhA polypeptide constant (C) regions as describedin FIGS. 1 and 2. The isolated nucleic acid may further encode one ormore non-conserved (V region) amino acids such as also identified inFIGS. 1 and 2.

Particular embodiments of such isolated nucleic acids are provided inSEQ ID NOS:28-32 and FIGS. 5-9.

The term “nucleic acid” as used herein designates single- ordouble-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA andgenomic DNA.

A “polynucleotide” is a nucleic acid having eighty (80) or morecontiguous nucleotides, while an “oligonucleotide” has less than eighty(80) contiguous nucleotides.

A “probe” may be a single or double-stranded oligonucleotide orpolynucleotide, suitably labeled for the purpose of detectingcomplementary sequences in Northern or Southern blotting, for example.

A “primer” is usually a single-stranded oligonucleotide, preferablyhaving 15-50 contiguous nucleotides, which is capable of annealing to acomplementary nucleic acid “template” and being extended in atemplate-dependent fashion by the action of a DNA polymerase such as Taqpolymerase, RNA-dependent DNA polymerase or Sequenase™.

The present invention also contemplates homologs of nucleic acids of theinvention as hereinbefore defined.

Such nucleic acid homologs exclude nucleic acids encoding full-lengthwild-type NhhA polypeptides.

For example, nucleic acid homologs encode peptides and polypeptides,structurally related to NhhA V and C regions of the invention, that maybe useful for the purposes of providing cross-protective immunity to N.meningitidis by immunization.

In one embodiment, nucleic acid homologs encode polypeptide homologs ofthe invention, inclusive of variants, fragments and derivatives thereof.

In another embodiment, nucleic acid homologs share at least 60%,preferably at least 70%, more preferably at least 80%, and even morepreferably at least 90% sequence identity with the nucleic acids of theinvention.

In yet another embodiment, nucleic acid homologs hybridize to nucleicacids of the invention under at least low stringency conditions,preferably under at least medium stringency conditions and morepreferably under high stringency conditions.

“Hybridize and Hybridization” is used herein to denote the pairing of atleast partly complementary nucleotide sequences to produce a DNA-DNA,RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementarynucleotide sequences occur through base-pairing between complementarypurines and pyrimidines as are well known in the art.

In this regard, it will be appreciated that modified purines (forexample, inosine, methylinosine and methyladenosine) and modifiedpyrimidines (thiouridine and methylcytosine) may also engage in basepairing.

“Stringency” as used herein, refers to temperature and ionic strengthconditions, and presence or absence of certain organic solvents and/ordetergents during hybridisation. The higher the stringency, the higherwill be the required level of complementarity between hybridizingnucleotide sequences.

“Stringent conditions” designates those conditions under which onlynucleic acid having a high frequency of complementary bases willhybridize.

Reference herein to low stringency conditions includes and encompasses:—

-   -   (i) from at least about 1% v/v to at least about 15% v/v        formamide and from at least about 1 M to at least about 2 M salt        for hybridisation at 42° C., and at least about 1 M to at least        about 2 M salt for washing at 42° C.; and    -   (ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH        7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1%        SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS        for washing at room temperature.

Medium stringency conditions include and encompass:—

-   -   (i) from at least about 16% v/v to at least about 30% v/v        formamide and from at least about 0.5 M to at least about 0.9 M        salt for hybridisation at 42° C., and at least about 0.5 M to at        least about 0.9 M salt for washing at 42° C.; and    -   (ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH        7.2), 7% SDS for hybridization at 65° C. and (a) 2×SSC, 0.1%        SDS; or (b) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS        for washing at 42° C.

High stringency conditions include and encompass:—

-   -   (i) from at least about 31% v/v to at least about 50% v/v        formamide and from at least about 0.01 M to at least about 0.15        M salt for hybridisation at 42° C., and at least about 0.01 M to        at least about 0.15 M salt for washing at 42° C.;    -   (ii) 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for        hybridization at 65° C., and (a) 0.1×SSC, 0.1% SDS; or (b) 0.5%        BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a        temperature in excess of 65° C. for about one hour; and    -   (iii) 0.2×SSC, 0.1% SDS for washing at or above 68° C. for about        20 minutes.

In general, washing is carried out at T_(m)=69.3+0.41 (G+C) %-12° C. Ingeneral, the T_(m) of a duplex DNA decreases by about 1° C. with everyincrease of 1% in the number of mismatched bases.

Notwithstanding the above, stringent conditions are well known in theart, such as described in Chapters 2.9 and 2.10 of. Ausubel et al.,supra, which are herein incorporated by reference. A skilled addresseewill also recognize that various factors can be manipulated to optimizethe specificity of the hybridization. Optimization of the stringency ofthe final washes can serve to ensure a high degree of hybridization.

Typically, complementary nucleotide sequences are identified by blottingtechniques that include a step whereby nucleotides are immobilized on amatrix (preferably a synthetic membrane such as nitrocellulose), ahybridization step, and a detection step. Southern blotting is used toidentify a complementary DNA sequence; northern blotting is used toidentify a complementary RNA sequence. Dot blotting and slot blottingcan be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNApolynucleotide sequences. Such techniques are well known by thoseskilled in the art, and have been described in Ausubel et al., supra, atpages 2.9.1 through 2.9.20. According to such methods, Southern blottinginvolves separating DNA molecules according to size by gelelectrophoresis, transferring the size-separated DNA to a syntheticmembrane, and hybridizing the membrane bound DNA to a complementarynucleotide sequence.

In dot blotting and slot blotting, DNA samples are directly applied to asynthetic membrane prior to hybridization as above.

An alternative blotting step is used when identifying complementarynucleic acids in a cDNA or genomic DNA library, such as through theprocess of plaque or colony hybridization. Other typical examples ofthis procedure is described in Chapters 8-12 of Sambrook et al., suprawhich are herein incorporated by reference.

Typically, the following general procedure can be used to determinehybridization conditions. Nucleic acids are blotted/transferred to asynthetic membrane, as described above. A wild type nucleotide sequenceof the invention is labeled as described above, and the ability of thislabeled nucleic acid to hybridize with an immobilized nucleotidesequence analyzed.

A skilled addressee will recognize that a number of factors influencehybridization. The specific activity of radioactively labeledpolynucleotide sequence should typically be greater than or equal toabout 10⁸ dpm/μg to provide a detectable signal. A radiolabelednucleotide sequence of specific activity 10⁸ to 10⁹ dpm/μg can detectapproximately 0.5 pg of DNA. It is well known in the art that sufficientDNA must be immobilized on the membrane to permit detection. It isdesirable to have excess immobilized DNA, usually 1-10 μg. Adding aninert polymer such as 10% (w/v) dextran sulfate (MW 500,000) orpolyethylene glycol 6000 during hybridization can also increase thesensitivity of hybridization (see Ausubel et al., supra at 2.10.10).

To achieve meaningful results from hybridization between a nucleic acidimmobilized on a membrane and a labeled nucleic acid, a sufficientamount of the labeled nucleic acid must be hybridized to the immobilizednucleic acid following washing. Washing ensures that the labeled nucleicacid is hybridized only to the immobilized nucleic acid with a desireddegree of complementarity to the labeled nucleic acid.

Methods for detecting labeled nucleic acids hybridized to an immobilizednucleic acid are well known to practitioners in the art. Such methodsinclude autoradiography, chemiluminescent, fluorescent and colorimetricdetection.

In another embodiment, nucleic acid homologs of the invention may beprepared according to the following procedure:

-   -   (i) obtaining a nucleic acid extract from a suitable host;    -   (ii) creating primers which are optionally degenerate wherein        each comprises a portion of a nucleotide sequence of the        invention; and    -   (iii) using said primers to amplify, via nucleic acid        amplification techniques, one or more amplification products        from said nucleic acid extract.

Suitably, the host is a bacterium.

Preferably, the host is of the genus Neisseria.

More preferably, the host is N. meningitidis or N. lactamica.

Primers useful according to nucleic acid sequence amplification methodsinclude SEQ ID NOS:40-51 as described in detail hereinafter.

Suitable nucleic acid amplification techniques are well known to theskilled addressee, and include polymerase chain reaction (PCR) as forexample described in Chapter 15 of Ausubel et al. supra, which isincorporated herein by reference; strand displacement amplification(SDA) as for example described in U.S. Pat. No. 5,422,252 which isincorporated herein by reference; rolling circle replication (RCR) asfor example described in Liu et al., 1996, J. Am. Chem. Soc. 118 1587and International application WO 92/01813 and Lizardi et al.,(International Application WO 97/19193) which are incorporated herein byreference; nucleic acid sequence-based amplification (NASBA) as forexample described by Sooknanan et al., 1994, Biotechniques 17 1077)which is incorporated herein by reference; and Q-β replicaseamplification as for example described by Tyagi et al., 1996, Proc.Natl. Acad. Sci. USA 93 5395 which is incorporated herein by reference.

As used herein, an “amplification product” refers to a nucleic acidproduct generated by nucleic acid amplification techniques.

Antibodies

The invention also contemplates antibodies against the isolated proteinsfragments, variants and derivatives of the invention. Antibodies of theinvention may be polyclonal or monoclonal. Well-known protocolsapplicable to antibody production, purification and use may be found,for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS INIMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D.Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring HarborLaboratory, 1988, which are both herein incorporated by reference.

Generally, antibodies of the invention bind to or conjugate with apolypeptide, fragment, variant or derivative of the invention. Forexample, the antibodies may comprise polyclonal antibodies. Suchantibodies may be prepared for example by injecting a polypeptide,fragment, variant or derivative of the invention into a productionspecies, which may include mice or rabbits, to obtain polyclonalantisera. Methods of producing polyclonal antibodies are well known tothose skilled in the art. Exemplary protocols which may be used aredescribed for example in Coligan et al., CURRENT PROTOCOLS INIMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.

In lieu of the polyclonal antisera obtained in the production species,monoclonal antibodies may be produced using the standard method as forexample, described in an article by Köhler & Milstein, 1975, Nature 256,495, which is herein incorporated by reference, or by more recentmodifications thereof as for example, described in Coligan et al.,CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or otherantibody producing cells derived from a production species which hasbeen inoculated with one or more of the polypeptides, fragments,variants or derivatives of the invention.

The invention also includes within its scope antibodies which compriseFc or Fab fragments of the polyclonal or monoclonal antibodies referredto above. Alternatively, the antibodies may comprise single chain Fvantibodies (scFvs) against the peptides of the invention. Such scFvs maybe prepared, for example, in accordance with the methods describedrespectively in U.S. Pat. No. 5,091,513, European Patent No 239,400 orthe article by Winter & Milstein, 1991, Nature 349 293, which areincorporated herein by reference.

The antibodies of the invention may be used for affinity chromatographyin isolating natural or recombinant N. meningitidis polypeptides. Forexample reference may be made to immunoaffinity chromatographicprocedures described in Chapter 9.5 of Coligan et al., CURRENT PROTOCOLSIN IMMUNOLOGY, supra.

The antibodies may be used to:

-   -   (i) screen expression libraries to identify variant polypeptides        of the invention;    -   (ii) identify immunoreactive fragments or immunoreactive        epitopes; and/or    -   (iii) detect N. meningitidis infection;        as will be described hereinafter but without limitation to these        particular uses.        Detection of N. meningitidis

The presence or absence of N. meningitidis in an individual may bedetermined by isolating a biological sample from said individual, mixingan antibody or antibody fragment described above with the biologicalsample, and detecting specifically bound antibody or antibody fragmentwhich indicates the presence of N. meningitidis in the sample.

The term “biological sample” as used herein refers to a sample that maybe extracted, untreated, treated, diluted or concentrated from anindividual, such as a patient. Suitably, the biological sample isselected from the group consisting of whole blood, serum, plasma,saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid,amniotic fluid, cerebrospinal fluid, skin biopsy, and the like.

Any suitable technique for determining formation of the complex may beused. For example, an antibody or antibody fragment according to theinvention having a label associated therewith may be utilized inimmunoassays. Such immunoassays may include, but are not limited to,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs)and immunochromatographic techniques (ICTs) which are well known thoseof skill in the art.

For example, reference may be made to Chapter 7 of Coligan et al.,CURRENT PROTOCOLS IN IMMUNOLOGY, supra which discloses a variety ofimmunoassays that may be used in accordance with the present invention.Immunoassays may include competitive assays as understood in the art.

The label associated with the antibody or antibody fragment may includethe following:

-   -   (A) direct attachment of the label to the antibody or antibody        fragment;    -   (B) indirect attachment of the label to the antibody or antibody        fragment; i.e., attachment of the label to another assay reagent        which subsequently binds to the antibody or antibody fragment;        and

(C) attachment to a subsequent reaction product of the antibody orantibody fragment.

The label may be selected from a group including a chromogen, acatalyst, an enzyme, a fluorophore, a chemiluminescent molecule, alanthanide ion such as Europium (Eu³⁴), a radioisotope and a directvisual label. In the case of a direct visual label, use may be made of acolloidal metallic or non-metallic particle, a dye particle, an enzymeor a substrate, an organic polymer, a latex particle, a liposome, orother vesicle containing a signal producing substance and the like.

A large number of enzymes useful as labels is disclosed in U.S. Pat. No.4,366,241, U.S. Pat. No. 4,843,000, and U.S. Pat. No. 4,849,338, all ofwhich are herein incorporated by reference. Enzyme labels useful in thepresent invention include alkaline phosphatase, horseradish peroxidase,luciferase, β-galactosidase, glucose oxidase, lysozyme, malatedehydrogenase and the like. The enzyme label may be used alone or incombination with a second enzyme in solution.

Suitably, the fluorophore is selected from a group including fluoresceinisothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITL) orR-Phycoerythrin (RPE).

The invention also extends to a method for detecting infection ofpatients by N. meningitidis, said method comprising the steps ofcontacting a biological sample from a patient with a polypeptide,fragment, variant or derivative of the invention, and determining thepresence or absence of a complex between said polypeptide, fragment,variant or derivative and N. meningitidis-specific antibodies in saidserum, wherein the presence of said complex is indicative of saidinfection.

In a preferred embodiment, detection of the above complex is effected bydetectably modifying said polypeptide, fragment, variant or derivativewith a suitable label as is well known in the art and using suchmodified compound in an immunoassay as for example described above.

In another aspect, the invention provides a method of detecting N.meningitidis bacteria in a biological sample suspected of containingsaid bacteria, said method comprising the steps of isolating thebiological sample from a patient, detecting a nucleic acid sequenceaccording to the invention in said sample which indicates the presenceof said bacteria. Detection of the said nucleic acid sequence may bedetermined using any suitable technique. For example, a labeled nucleicacid according to the invention may be used as a probe in a Southernblot of a nucleic acid extract obtained from a patient as is well knownin the art.

Alternatively, a labeled nucleic acid according to the invention may beutilized as a probe in a Northern blot of a RNA extract from thepatient.

Preferably, a nucleic acid extract from the patient is utilized inconcert with oligonucleotide primers corresponding to sense andantisense sequences of a nucleic acid sequence according to theinvention, or flanking sequences thereof, in a nucleic acidamplification reaction such as PCR, or the ligase chain reaction (LCR)as for example described in International Application WO89/09385 whichis incorporated by reference herein.

A variety of automated solid-phase detection techniques are alsoappropriate. For example, very large scale immobilized primer arrays(VLSIPS™) are used for the detection of nucleic acids as for exampledescribed by Fodor et al., 1991, Science 251 767 and Kazal et al., 1996,Nature Medicine 2 753. The above generic techniques are well known topersons skilled in the art.

Pharmaceutical Compositions

A further feature of the invention is the use of the polypeptide,fragment, variant or derivative of the invention (“immunogenic agents”)as actives in a pharmaceutical composition for protecting patientsagainst infection by N. meningitidis.

Suitably, the pharmaceutical composition comprises apharmaceutically-acceptable carrier, diluent or excipient.

By “pharmaceutically-acceptable carrier, diluent or excipient” is meanta solid or liquid filler, diluent or encapsulating substance that may besafely used in systemic administration. Depending upon the particularroute of administration, a variety of carriers, well known in the artmay be used. These carriers may be selected from a group includingsugars, starches, cellulose and its derivatives, malt, gelatine, talc,calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffered solutions, emulsifiers, isotonic saline and saltssuch as mineral acid salts including hydrochlorides, bromides andsulfates, organic acids such as acetates, propionates and malonates andpyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers,diluents and excipients is Remington's Pharmaceutical Sciences (MackPublishing Co. N.J. USA, 1991) which is incorporated herein byreference.

Any safe route of administration may be employed for providing a patientwith the composition of the invention. For example, oral, rectal,parenteral, sublingual, buccal, intravenous, intra-articular,intramuscular, intra-dermal, subcutaneous, inhalational, intraocular,intraperitoneal, intracerebroventricular, transdermal and the like maybe employed. Intra-muscular and subcutaneous injection is appropriate,for example, for administration of immunogenic compositions, vaccinesand DNA vaccines.

Dosage forms include tablets, dispersions, suspensions, injections,solutions, syrups, troches, capsules, suppositories, aerosols,transdermal patches and the like. These dosage forms may also includeinjecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of the therapeuticagent may be effected by coating the same, for example, with hydrophobicpolymers including acrylic resins, waxes, higher aliphatic alcohols,polylactic and polyglycolic acids and certain cellulose derivatives suchas hydroxypropylmethyl cellulose. In addition, the controlled releasemay be effected by using other polymer matrices, liposomes and/ormicrospheres.

Pharmaceutical compositions of the present invention suitable for oralor parenteral administration may be presented as discrete units such ascapsules, sachets or tablets each containing a pre-determined amount ofone or more therapeutic agents of the invention, as a powder or granulesor as a solution or a suspension in an aqueous liquid, a non-aqueousliquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Suchcompositions may be prepared by any of the methods of pharmacy but allmethods include the step of bringing into association one or moreimmunogenic agents as described above with the carrier which constitutesone or more necessary ingredients. In general, the compositions areprepared by uniformly and intimately admixing the immunogenic agents ofthe invention with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product into the desiredpresentation.

The above compositions may be administered in a manner compatible withthe dosage formulation, and in such amount as isimmunogenically-effective to protect patients from N. meningitidisinfection. The dose administered to a patient, in the context of thepresent invention, should be sufficient to effect a beneficial responsein a patient over time such as a reduction in the level of N.meningitidis, or to inhibit infection by N. meningitidis. The quantityof the immunogenic agent(s) to be administered may depend on the subjectto be treated inclusive of the age, sex, weight and general healthcondition thereof. In this regard, precise amounts of the immunogenicagent(s) required to be administered will depend on the judgement of thepractitioner.

In determining the effective amount of the immunogenic agent to beadministered in the treatment or prophylaxis against N. meningitidis,the physician may evaluate circulating plasma levels, progression ofdisease, and the production of anti-N. meningitidis antibodies. In anyevent, suitable dosages of the immunogenic agents of the invention maybe readily determined by those of skill in the art. Such dosages may bein the order of nanograms to milligrams of the immunogenic agents of theinvention.

The above compositions may be used as therapeutic or prophylacticvaccines. Accordingly, the invention extends to the production ofvaccines containing as actives one or more of the immunogenic agents ofthe invention. A variety of applicable procedures are contemplated forproducing such vaccines. Exemplary procedures include, for example,those described in NEW GENERATION VACCINES (1997, Levine et al., MarcelDekker, Inc. New York, Basel Hong Kong) which is incorporated herein byreference.

An immunogenic agent according to the invention can be mixed, conjugatedor fused with other antigens, including B or T cell epitopes of otherantigens. In addition, it can be conjugated to a carrier as describedbelow.

When an haptenic peptide of the invention is used (i.e., a peptide whichreacts with cognate antibodies, but cannot itself elicit an immuneresponse), it can be conjugated with an immunogenic carrier. Usefulcarriers are well known in the art and include for example:thyroglobulin; albumins such as human serum albumin; toxins, toxoids orany mutant crossreactive material (CRM) of the toxin from tetanus,diptheria, pertussis, Pseudomonas, E. coli, Staphylococcus, andStreptococcus; polyamino acids such as poly(lysine:glutamic acid);influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus coreprotein; hepatitis B virus recombinant vaccine and the like.Alternatively, a fragment or epitope of a carrier protein or otherimmunogenic protein may be used. For example, a haptenic peptide of theinvention can be coupled to a T cell epitope of a bacterial toxin,toxoid or CRM. In this regard, reference may be made to U.S. Pat. No.5,785,973 which is incorporated herein by reference.

In addition, a polypeptide, fragment, variant or derivative of theinvention may act as a carrier protein in vaccine compositions directedagainst Neisseria, or against other bacteria or viruses.

The immunogenic agents of the invention may be administered asmultivalent subunit vaccines in combination with antigens of N.meningitidis, or antigens of other organisms inclusive of the pathogenicbacteria H. influenzae, M. catarrhalis, N. gonorrhoeae, E. coli, S.pneumoniae etc. Alternatively or additionally, they may be administeredin concert with oligosaccharide or polysaccharide components of N.meningitidis.

The vaccines can also contain a pharmaceutically-acceptable carrier,diluent or excipient as hereinbefore defined.

The vaccines and immunogenic compositions may include an adjuvant as iswell known in the art. Adjuvants contemplated by the present inventioninclude, but are not limited to: surface active substances such ashexadecylamine, octadecylamine, octadecyl amino acid esters,lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N′,N′bis(2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol, andpluronic polyols; polyamines such as pyran, dextransulfate, poly ICcarbopol; peptides such as muramyl dipeptide and derivatives,dimethylglycine, tuftsin; oil emulsions; and mineral gels such asaluminum phosphate, aluminum hydroxide or alum; lymphokines, QuilA andimmune stimulating complexes (ISCOMS).

With regard to examples of adjuvants, reference is also made toInternational Publication WO99/36544 incorporated herein by reference.

Vaccination by DNA Delivery

Expression constructs comprising modified NhhA proteins of the inventionmay be administered to humans to prophylactically and/or therapeuticallytreat the host. In this regard, expression constructs may encode one ormore modified NhhA peptides, polypeptides, fragments or derivatives ofthese, collectively referred to as “immunogenic agents”.

Expression constructs also include gene therapy constructs, which employspecialized gene therapy vectors such as vaccinia, and viral vectorsuseful in gene therapy. The latter include adenovirus andadenovirus-associated viruses (AAV) such as described in Franceschi etal., 2000, J. Cell Biochem. 78:476, Braun-Falco et al., 1999, Gene Ther.6:432, retroviral and lentiviral vectors such as described inBuchshacher et al., 2000, Blood 95:2499 and vectors derived from herpessimplex virus and cytomegalovirus. A general review of gene therapyvectors and delivery methods may be found in Robbins et al., 1998,Trends in Biotech. 16:35. An exemplary reference which describes anumber of vectors potentially suitable for gene therapy using Neisseriaproteins, and methods of delivery, is International PublicationWO99/36544 incorporated herein by reference.

The immunogenic agents of the invention may be expressed by attenuatedviral hosts. By “attenuated viral hosts” is meant viral vectors that areeither naturally, or have been rendered, substantially avirulent. Avirus may be rendered substantially avirulent by any suitable physical(e.g., heat treatment) or chemical means (e.g., formaldehyde treatment).By “substantially avirulent” is meant a virus whose infectivity has beendestroyed. Ideally, the infectivity of the virus is destroyed withoutaffecting the proteins that carry the immunogenicity of the virus. Fromthe foregoing, it will be appreciated that attenuated viral hosts maycomprise live viruses or inactivated viruses.

Attenuated viral hosts which may be useful in a vaccine according to theinvention may comprise viral vectors inclusive of adenovirus,cytomegalovirus and preferably pox viruses such as vaccinia (see forexample Paoletti and Panicali, U.S. Pat. No. 4,603,112 which isincorporated herein by reference) and attenuated Salmonella strains (seefor example Stocker, U.S. Pat. No. 4,550,081 which is hereinincorporated by reference). Live vaccines are particularly advantageousbecause they lead to a prolonged stimulus that can confer substantiallylong-lasting immunity. Another reference which describes a variety ofviral vectors potentially suitable for immunization using Neisseriaproteins, and methods of delivery, is International PublicationWO99/36544 incorporated herein by reference.

Multivalent vaccines can be prepared from one or more microorganismsthat express different epitopes of N. meningitidis (e.g., other surfaceproteins or epitopes of N. meningitidis). In addition, epitopes of otherpathogenic microorganisms can be incorporated into the vaccine.

In a preferred embodiment, this will involve the construction of arecombinant vaccinia virus to express a nucleic acid sequence accordingto the invention. Upon introduction into a host, the recombinantvaccinia virus expresses the immunogenic agent, and thereby elicits ahost CTL response. For example, reference may be made to U.S. Pat. No.4,722,848, incorporated herein by reference, which describes vacciniavectors and methods useful in immunization protocols.

A wide variety of other vectors useful for therapeutic administration orimmunization with the immunogenic agents of the invention will beapparent to those skilled in the art from the present disclosure.

In a further embodiment, the nucleotide sequence may be used as avaccine in the form of a “naked DNA” vaccine as is known in the art. Forexample, an expression vector of the invention may be introduced into amammal, where it causes production of a polypeptide in vivo, againstwhich the host mounts an immune response as for example described inBarry, M. et al., (1995, Nature, 377:632-635) which is herebyincorporated herein by reference.

Detection Kits

The present invention also provides kits for the detection of N.meningitidis in a biological sample. These will contain one or moreparticular agents described above depending upon the nature of the testmethod employed. In this regard, the kits may include one or more of apolypeptide, fragment, variant, derivative, antibody, antibody fragmentor nucleic acid according to the invention. The kits may also optionallyinclude appropriate reagents for detection of labels, positive andnegative controls, washing solutions, dilution buffers and the like. Forexample, a nucleic acid-based detection kit may include (i) a nucleicacid according to the invention (which may be used as a positivecontrol), (ii) an oligonucleotide primer according to the invention, andoptionally a DNA polymerase, DNA ligase etc depending on the nucleicacid amplification technique employed.

Preparation of Immunoreactive Fragments

The invention also extends to a method of identifying an immunoreactivefragment of a polypeptide, variant or derivatives according to theinvention. This method essentially comprises generating a fragment ofthe polypeptide, variant or derivative, administering the fragment to amammal; and detecting an immune response in the mammal. Such responsewill include production of elements which specifically bind N.meningitidis and/or said polypeptide, variant or derivative, and/or aprotective effect against N. meningitidis infection.

Prior to testing a particular fragment for immunoreactivity in the abovemethod, a variety of predictive methods may be used to deduce whether aparticular fragment can be used to obtain an antibody that cross-reactswith the native antigen. These predictive methods may be based onamino-terminal or carboxy-terminal sequence as for example described inChapter 11.14 of Ausubel et al., supra. Alternatively, these predictivemethods may be based on predictions of hydrophilicity as for exampledescribed by Kyte & Doolittle 1982, J. Mol. Biol. 157 105 and Hopp &Woods, 1983, Mol. Immunol. 20 483) which are incorporated by referenceherein, or predictions of secondary structure as for example describedby Choo & Fasman, 1978, Ann. Rev. Biochem. 47 251), which isincorporated herein by reference.

In addition, “epitope mapping” uses monoclonal antibodies of theinvention to identify cross-reactive epitopes by first testing theirability to provide cross-protection, followed by identifying the epitoperecognized by said antibodies. An exemplary method is provided inColigan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra.

Generally, peptide fragments consisting of 10 to 15 residues provideoptimal results. Peptides as small as 6 or as large as 20 residues haveworked successfully. Such peptide fragments may then be chemicallycoupled to a carrier molecule such as keyhole limpet hemocyanin (KLH) orbovine serum albumin (BSA) as for example described in Sections 11.14and 11.15 of Ausubel et al., supra).

It will also be appreciated that peptides may be syntheticallycircularized, as for example described in Hoogerhout et al., 1995,Infect. Immun. 63 3473, which is herein incorporated by reference.

The peptides may be used to immunize an animal as for example discussedabove. Antibody titers against the native or parent polypeptide fromwhich the peptide was selected may then be determined by, for example,radioimmunoassay or ELISA as for instance described in Sections 11.16and 114 of Ausubel et al., supra.

Antibodies may then be purified from a relevant biological fluid of theanimal by ammonium sulfate fractionation or by chromatography as is wellknown in the art. Exemplary protocols for antibody purification aregiven in Sections 10.11 and 11.13 of Ausubel et al., supra, which areherein incorporated by reference.

Immunoreactivity of the antibody against the native or parentpolypeptide may be determined by any relevant procedure such as, forexample, Western blot.

Functional Blockers

The wild-type NhhA/HiaNm polypeptides disclosed in WO99/31132 arebelieved to have adhesin properties. They in fact have some similarityto adhesins of Haemophilus influenzae which are surface antigens.Specifically they are approximately 67% homologous to the Hia protein ofH. influenzae (Barenkamp & St. Geme III, 1996, Molecular Microbiology 191215), and 74% homologous to the Hsf protein of H. influenzae (St. GemeIII, J. et al, 1996, Journal of Bacteriology 178 6281; and U.S. Pat. No.5,646,259). For these comparisons, a gap weight of 3, and length weightof 0.01 was used using the GAP program (Deveraux, 1984, supra). Thus,interruption of the function of these polypeptides would be ofsignificant therapeutic benefit since they would prevent N. meningitidisbacteria from adhering to and invading cells. Interruption of thefunction may be effected in several ways.

For example, moieties such as chemical reagents or polypeptides whichblock receptors on the cell surface which interact with a polypeptidesof the invention may be administered. These compete with the infectiveorganism for receptor sites. Such moieties may comprise for examplepolypeptides of the invention, in particular fragments, or functionalequivalents of these as well as mimetics.

The term “mimetics” is used herein to refer to chemicals that aredesigned to resemble particular functional regions of the proteins orpeptides. Anti-idiotypic antibodies raised against the above-describedantibodies which block the binding of the bacteria to a cell surface mayalso be used. Alternatively, moieties which interact with the receptorbinding sites in the polypeptides of the invention may effectivelyprevent infection of a cell by N. meningitidis. Such moieties maycomprise blocking antibodies, peptides or other chemical reagents.

All such moieties, pharmaceutical compositions in which they arecombined with pharmaceutically acceptable carriers and methods oftreating patients suffering from N. meningitidis infection byadministration of such moieties or compositions form a further aspect ofthe invention.

The polypeptides of the invention may be used in the screening ofcompounds for their use in the above methods. For example, polypeptidesof the invention may be combined with a label and exposed to a cellculture in the presence of a reagent under test. The ability of reagentto inhibit the binding of the labeled polypeptide to the cell surfacecan then be observed. In such a screen, the labeled polypeptides may beused directly on an organism such as E. coli. Alternatively, N.meningitidis itself may be engineered to express a modified anddetectable form of the polypeptide. The use of engineered N.meningitidis strains in this method is preferred as it is more likelythat the tertiary structure of the protein will resemble more closelythat expressed in wild-type bacteria.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLE 1 Identification of Constant and Variable Regions of NhhAPolypeptides

The present inventors have elucidated NhhA amino acid sequences whichare conserved and/or non-conserved between ten (10) strains of N.meningitidis. The non-conserved regions are subdivided into fourvariable regions (V1, V2, V3 and V4) and the conserved regions aresubdivided into C1, C2, C3, C4 and C5 (as shown in FIG. 1 and Table 1;SEQ ID NOS:1-11). The corresponding nucleotide sequence comparison isshown in FIG. 2 (SEQ ID NOS:12-22).

EXAMPLE 2 PMC 21 NhhA Polypeptide Over-Expression

The NhhA protein encoded by the nhhA gene of N. meningitidis strainPMC21 was over expressed by making an expression construct wherein thenhhA gene is operably linked to a promoter.

The following oligonucleotide primers were used to amplify an N.meningitidis PMC21 strain nhhA nucleic acid open reading frame by PCR:—

(SEQ ID NO 40) HOMP5′: 5′-CAA TTA ACG GCC GAA TAA AAG GAA GCCGAT ATG AAC AAA ATA TAC CGC ATC-3′;which contains an EagI restriction site(underlined) and the sequence encoding thefirst 7 (seven) amino acids of NhhA (bold type) (SEQ ID NO 41) HOMP3′AN5′-TGG AAT CCA TGG AAT CGC CAC CCT TCC CTT C-3′;which contains an NcoI restriction site(underlined) and the reverse complementof sequence 48-61 nucleotides past the endof the nhhA open reading frame of strain ¢3 (bold type)

The amplification product contained restriction sites which weresubsequently digested with EagI and NcoI restriction endonucleases.

The plasmid used for subcloning was pCO14K, which plasmid contains aporA promoter upstream of the gene encoding the strongly expressed Class1 outer membrane protein of N. meningitidis together with flankingsequence of N. meningitidis strain 2996 and a selectable kanamycinresistance gene as described by Rouppe van der Voort, et al., InfectImmun 1996 64:2745.

The digested amplification product was then ligated into EagI and NcoIrestriction endonuclease-digested pCO14K. This ligation resulted in thereplacement of the majority of the porA open reading frame with the nhhAamplification product (FIG. 3). This created a recombinant nucleic acidexpression construct (open reading frame shown in SEQ ID NO:12) whichencodes a polypeptide of 591 amino acids as shown in SEQ ID NO:1.

This places expression of the nhhA nucleic acid of the invention underthe control of the strong porA promoter. Translation begins at the ATGcodon beginning at position 31 of HOMP5′. In order to prevent formationof a fusion between the porA and nhhA, the HOMP5′ sequence contains aTAA stop codon prior to the initiating ATG described above.

The resulting plasmid, pIP52(PMC21), was linearized by restrictiondigestion and used to transform N. meningitidis strain 7G2 using themethod described by Janik et al, 1976, Journal of Clinical Microbiology4 71. Transformants were selected by overnight incubation at 37° C. in5% CO₂ on solid media containing 100 μg/ml kanamycin. Kanamycinresistant colonies were selected, subcultured overnight and screened forover-expression of NhhA polypeptide by separating total cell proteinselectrophoretically on 10% SDS-PAGE followed by transfer tonitrocellulose membrane using a Semi-Dry Blotter (BioRad). The membranewas then incubated sequentially with rabbit anti-NhhA sera (as describedin International Publication WO99/31132) and alkaline-phosphataseconjugated anti-Rabbit IgG (Sigma) before colorimetric detection withNBT/BCIP (Sigma). One clone was isolated which expressed NhhApolypeptide at a higher level compared with the parental strain (FIG.11). Analysis of the predicted amino acid sequence using the computerprogram SIGCLEAVE (part of the eGCG suite of programs available from theAustralian National Genomic Information Service {ANGIS}) indicates thatthe first 51 amino acids will be cleaved to produce the maturepolypeptide (FIG. 14A; SEQ ID NO:33).

The plasmid construct pIP52(PMC21) may be transformed into anytransformation-competent strain of N. meningitidis.

EXAMPLE 3 H41 NhhA Polypeptide Over-Expression

The NhhA protein encoded by the nhhA gene of N. meningitidis strain H41was over expressed using the same methods as described in Example 2.This created a recombinant nucleic acid expression construct (openreading frame shown in SEQ ID NO:13) which encodes a polypeptide of 591amino acids as shown in SEQ ID NO:2. In this example the resultingplasmid pIP52(H41) was linearized, and transformed into N. meningitidisstrain 7G2. Kanamycin resistant colonies were analysed and one waschosen which when examined by Western immunoblot, demonstratedoverexpression of NhhA. (FIG. 11). Analysis of the predicted amino acidsequence using the computer program SIGCLEAVE (part of the eGCG suite ofprograms available from the Australian National Genomic InformationService {ANGIS}) indicates that the first 51 amino acids will be cleavedto produce the mature polypeptide (FIG. 14B; SEQ ID NO:34).

This strategy may be employed to create expression constructs containingthe wild-type nhhA sequence of other N. meningitidis strains.

EXAMPLE 4 NhhA Deletion Mutant Construction Using Convenient RestrictionSite

For ease of reference, the amino acid sequence of the NhhA polypeptideencoded by the nhhA nucleic acid of strain PMC21 is shown in SEQ IDNO 1. The present inventors created a deletion mutant version ofwild-type PMC21 nhhA, in which the most variable region between strainswas deleted. An amplification product encoding amino acids 1-54 of thewild-type PMC21 NhhA polypeptide was generated by PCR amplification fromnhhA nucleic acid template using the following primers:

(SEQ ID NO 40) HOMP5′: 5′-CAA TTA ACG GCC GAA TAA AAG GAA GCCGAT ATG AAC AAA ATA TAC CGC ATC-3′;which is the same oligonucleotide used tocreate the overexpression construct pIP52. (SEQ ID NO 42) NH3′BG:5′-GGT CAG ATC TGT TTC ATT GTT AGC ACT TGC-3′;which contains a BglII restriction site(underlined) and the reverse complementof sequence encoding amino acids 134,(double underlined) and 49-54 of wild-type PMC21 NhhA (bold type).

The resulting amplification product included an EagI and BglIIrestriction endonuclease sites. pIP52(PMC21) includes a single EagI site20 bp upstream of the start of the nhhA open reading frame (ORF) and asingle BglII site located within the ORF (see FIG. 3B). Therefore,pIP52(PMC21) and the amplification product were subjected to restrictionendonuclease digestion with EagI and BglII, ligated and used totransform competent DH5a strain E. coli bacteria; this replaces theEagI/BglII fragment of pIP52(PMC21) with the PCR product. This created arecombinant nucleic acid expression construct (open reading frame shownin FIG 5B; SEQ ID NO:28) which encodes a polypeptide of 512 amino acidsas shown in FIG 5A (SEQ ID NO:23). This amino acid sequence includesamino acids 1-54 and 134-592 of the wild-type sequence, and therebydeletes the majority of the V1 region, all of the V2 and C2 regions, andpart of the C3 region of the wild-type PMC21 NhhA polypeptide.

This plasmid was linearised by restriction digestion and transformed into N. meningitidis strain 7G2. Using methods as described in Example 1,one clone was isolated which overexpresses the truncated PMC21 NhhA(FIG. 11).

Analysis of the predicted amino acid sequence using the computer programSIGCLEAVE (part of the eGCG suite of programs available from theAustralian National Genomic Information Service {ANGIS}) indicates thatthe first 51 amino acids will be cleaved to produce the maturepolypeptide (FIG. 14C; SEQ ID NO:35). To confirm the presence of acleavable signal sequence and to confirm the identity of the overexpressed protein, outer membrane proteins were semi-purified byisolating the fraction that is insoluble in the detergent sarkosyl.

The isolated membrane proteins were separated electrophoretically beforetransfer to Nylon membrane. The position of the over expressed proteinwas revealed by Coomassie stain. This region of the membrane was excisedand the protein was N-terminal sequenced. The first eleven amino acidsof this protein were XXETDLTSVGT (SEQ ID NO:52) which corresponds toamino acid residues 52 to 62 (inclusive) of the amino acid sequencepredicted to be expressed by the over expression construct as defined inthis example.

This is an example of a deletion using existing restriction sites withinthe polynucleotide sequence. This construct may be transformed into anytransformation competent N. meningitidis.

EXAMPLE 5 NhhA Deletion Mutant Construction Using Convenient RestrictionSite

An expression construct containing the wild-type nhhA sequence of H41was made as described in Example 2. The resulting expression constructwas named pIP52(H41). A deletion mutant was made, using the strategyoutlined in this example. In this instance the oligonucleotide primersused were:

(SEQ ID NO: 40) HOMP5′: 5′-CAA TTA ACG GCC GAA TAA AAG GAA GCCGAT ATG AAC AAA ATA TAC CGC ATC-3′;which is the same oligonucleotide used tocreate the overexpression construct pIP52 (SEQ ID NO 43) NH3′STU:5′-GAT CAG GCC TGT ATC TTC ATC GGT AGC ATT-3′;which contains a StuI restriction site(underlined) and the reverse complementof sequence encoding amino acids 134,(double underlined) and 49-54 of wild-type H41 NhhA (bold type).

The resulting amplification product contains single EagI and StuIrestriction endonuclease sites. The expression construct pIP52(H41)contains these restriction sites. Therefore, pIP52(H41) and theamplification product were subjected to restriction endonucleasedigestion with EagI and StuI, ligated and used to transform competentDH5α strain E. coli bacteria; this ligation replaces the EagI/StuIfragment of pIP52(H41) with the PCR product. This created a recombinantnucleic acid expression construct (open reading frame shown in FIG. 6Band SEQ ID NO:29) which encodes a polypeptide of 513 amino acids asshown in FIG. 6A and SEQ ID NO:24. This amino acid sequence includesamino acids 1-54 and 134-593 of the wild-type sequence, and therebydeletes the majority of the V1 region, all of the V2 and C2 regions, andpart of the C3 region of the wild-type H41 NhhA polypeptide.

This plasmid was linearised by restriction digestion and transformed into N. meningitidis strain 7G2. Using methods as described in Example 1,one clone was isolated which overexpresses the truncated H41 NhhA (FIG.11).

Analysis of the predicted amino acid sequence using the computer programSIGCLEAVE (part of the eGCG suite of programs available from theAustralian National Genomic Information Service {ANGIS}) indicates thatthe first 51 amino acids will be cleaved to produce the maturepolypeptide (FIG. 14D; SEQ ID NO:36).

In this example, a construct was made containing the C1 and C5 regions,and all other regions deleted (see FIG. 5A).

The following oligonucleotide primers were used in PCR reactions toamplify DNA corresponding to region C1 (see FIG. 1) from chromosomal DNAof strain PMC21:

(SEQ ID NO: 40) HOMP5′: 5′-CAA TTA ACG GCC GAA TAA AAG GAA GCCGAT ATG AAC AAA ATA TAC CGC ATC-3′;which is the same oligonucleotide used tocreate the over-expression construct pIP52(PMC21) (SEQ ID NO 44) SO-C:5′-GAC GAA ATC AAC GTT CTT AGC ACT TGC CTG AAC CGT TGC-3′;which sequence is the reverse complementof sequence encoding amino acids 237-241at the start of the C5 region (underlined)and amino acids 45-52 at the end of theC1 region (bold type) of wild-type NhhA of strain PMC21.The amplification product of this reaction is HOMP5′/SO-C.

The following oligonucleotide primers were used in PCR reactions toamplify C5 from chromosomal DNA of strain PMC21:

(SEQ ID NO 45) SO-D: 5′-AAC GTT GAT TTC GTC CGC ACT TAC-3′;which encodes amino acids 237-244 at thestart of C5 (underlined indicates reverse complement of Primer SO-C),(SEQ ID NO 41) HO3′AN: 5′-TGG AAT CCA TGG AAT CGC CAC CCT TCC CTT C-3′;which is the same primer used in the construction of pIP52.The amplification product of this reaction is SO-D/HO3'AN.

The amplification products HOMP5′/SO-C and SO-D/HO3′AN were purifiedfrom an agarose gel following separation by electrophoresis, were mixed,and subjected to further amplification using primers HOMP5′ and HO3′AN.The resulting amplification product encodes amino acids 1-52 and 337-591of wild-type NhhA of PMC21. This amplification product was subjected torestriction digestion with EagI and NcoI, and cloned into pCO14K, asdescribed in Example 1. This recombinant molecule contains regions C1and C5, thus deleting regions V1 to 4 and C2 to 4. The nucleotidesequence of the open reading frame is shown in FIG. 7B and SEQ ID NO:30,and the predicted polypeptide sequence derived from this nucleotidesequence is shown in FIG. 7A and SEQ ID NO:25.

This plasmid was linearized by restriction digestion and transformed into N. meningitidis strain 7G2. Using methods as described in Example 2,one clone was isolated which overexpresses the truncated PMC21 NhhA.

Analysis of the predicted amino acid sequence using the computer programSIGCLEAVE (part of the eGCG suite of programs available from theAustralian National Genomic Information Service {ANGIS}) indicates thatthe first 51 amino acids will be cleaved to produce the maturepolypeptide (FIG. 14E; SEQ ID NO:37).

This plasmid may be transformed into any transformation competent strainof N. meningitidis.

EXAMPLE 7 NhhA Deletion Mutant Construction Using Splice-Overlap PCR

It will be appreciated that a similar strategy can be used to createrecombinant polynucleotides encoding various regions of NhhA. Aconstruct can be made comprising regions C1, C4, V4 and C5 using thefollowing strategy (see FIG. 5B):

The C1 region is amplified using oligonucleotide primers:

(SEQ ID NO: 40) HOMP5′: 5′-CAA TTA ACG GCC GAA TAA AAG GAA GCCGAT ATG AAC AAA ATA TAC CGC ATC-3′; (SEQ ID NO: 46) SO-E:5′-AAC GCT TGC CGC ACG CTT AGC ACT TGC CTG CAA CGT TGC-3′;which encodes the reverse complement ofamino acids 211-215 at the start of the C4region (underlined) and at the end of theC1 region (bold type) of strain PMC21.The amplification product of this reaction is HOMP5′/SO-E.

The following oligonucleotide primers are used in PCR reactions toamplify the region C4-V4-C5 from chromosomal DNA of strain PMC21:

(SEQ ID NO: 47) SO-F: 5′-CGT GCG GCA AGC GTT AAA GAC GTA-3′;which encodes amino acids 211-218 at thestart of C4 (underlined indicates reverse complement of Primer SO-E),(SEQ ID NO: 41) HO3′AN: 5′-TGG AAT CCA TGG AAT CGC CAC CCT TCC CTT C-3.The amplification product of this reaction is SO-F/HOMP3′.

The amplification products HOMP5′/SO-E and SO-F/HO3′AN will be purifiedfrom agarose gel following separation by electrophoresis, and will bemixed, and subjected to further amplification using primers HOMP5′ andHO3′AN. The resulting product encodes amino acids 1-52 and 211-591 ofwild-type NhhA of PMC21. This amplification product will be subjected torestriction digestion with EagI and NcoI, and cloned into pCO14K. Thisrecombinant molecule contains regions C1, C4, V4 and C5 thus deletingregions V1-3 and C2-3. The nucleotide sequence of the open reading frameis shown in FIG. 8B and SEQ ID NO:31, and the predicted polypeptidesequence derived from this nucleotide sequence is shown in FIG. 8A andSEQ ID NO:26. Analysis of the predicted amino acid sequence using thecomputer program SIGCLEAVE (part of the eGCG suite of programs availablefrom the Australian National Genomic Information Service {ANGIS})indicates that the first 51 amino acids will be cleaved to produce themature polypeptide (FIG. 14F; SEQ ID NO:38).

EXAMPLE 8 NhhA Deletion Mutant Construction Using Splice-Overlap PCR

It will be appreciated that a similar strategy can be used to createrecombinant polynucleotides encoding various regions of NhhA. Aconstruct can be made comprising regions C1, C2, C3, C4, and C5 usingthe following strategy (see FIG. 5C):

C1 and C2 will be amplified using oligonucleotide primers:

(SEQ ID NO: 40) HOMP5′: 5′-CAA TTA ACG GCC GAA TAA AAG GAA GCCGAT ATG AAC AAA ATA TAC CGC ATC-3′; (SEQ ID NO: 48) SO-G:5′-CAG CGA GTA GGT GAA  TTG TTT GAT TTT CAG GTT GTC GCC GGC TTT GAG GGTGTT AGC ACT TGC CTG AAC CGT-3′; which encodes the reverse complement ofamino acids 125-129 at the start of the C3region (underlined), all of the C2 region(amino acids 109-120, bold and doubleunderlined) and the end of the C1 region(amino acids 46-52, bold type) of strain PMC21.The amplification product of this reaction is HOMP5′/SO-G.

The C3 and part of C4 regions will be amplified using the followingoligonucleotide primers:

(SEQ ID NO 49) SO-H: 5′-TTC ACC TAC TCG CTG AAA AAA GAC-3′;which encodes amino acids 125-132 at thestart of C3 (underlined indicates reverse complement of Primer SO-G)(SEQ ID NO: 50) SO-I: 5′-GCC AGC GTT TAA TAC GTC TTT AAC GCTTGC CGC ACG  ATC GGT CAA AGT CGA ACC AAT -3′;which encodes the reverse complement ofamino acids 182-88 at the end of C3 (under-lined) and amino acids 211-222 of C4 (bold type).The amplification product of this reaction is SO-H/SO-I.

The amplification products HOMP5′/SO-G and SO-H/SO-I are purified fromagarose gel following separation by electrophoresis, mixed and subjectedto further amplification using primers HOMP5′ and SO-I to yield aproduct encoding amino acids 1-52, 103-114, 125-188, and 211-222, i.e.regions C1, C2, C3 and part of C4. The amplification product of thisreaction is HOMP5′/SO-I.

The C5 and part of C4 regions are amplified using the followingoligonucleotide primers:

(SEQ ID NO: 51) SO-J: 5′ GTA TTA AAC GCT GGC  TGG AAC ATT AAAGGC GTT AAA AAC GTT GAT TTC GTC CGC ACT-3′;which encodes amino acids 218-229 of C4(underlined), and amino acids 237-243 ofC5 (bold type) of wild-type NhhA of strainPMC21. (Bold underlined type indicates re- verse complement of SO-I)(SEQ ID NO: 41) HO3′AN: 5′-TGG AAT CCA TGG AAT CGC CAC CCT TCC CTT C-3′.The amplification product of this reaction is SO-J/HO3'AN.

The amplification products HOMP5′/SO-I and SO-J/HO3′AN will be purifiedfrom agarose gel following separation by electrophoresis, and will bemixed, and subjected to further amplification using primers HOMP5′ andHO3′AN. The resulting product encodes amino acids 1-52, 103-114,125-188, 211-229, and 237-591 of wild-type NhhA of strain PMC21. Theresulting product will be subjected to restriction digestion with EagIand NcoI, and cloned into pCO14K. This recombinant molecule containsregions C1, C2, C3, C4 and C5, thus deleting regions V1, V2, V3, and V4.The nucleotide sequence of the open reading frame is shown in FIG. 9Band SEQ ID NO:32, and the predicted polypeptide sequence derived fromthis nucleotide sequence is shown in FIG. 9A and SEQ ID NO: 27. Analysisof the predicted amino acid sequence using the computer programSIGCLEAVE (part of the eGCG suite of available from the AustralianNational Genomic Information Service {ANGIS}) indicates that the first51 amino acids will be cleaved to produce the mature polypeptide (FIG.14G; SEQ ID NO:39).

This construct can be transformed into any transformation competentstrain of N. meningitidis.

EXAMPLE 9 Purification of Over Expressed NhhA Polypeptides

Recombinant NhhA polypeptide as described in the previous Examples maybe isolated by the following procedure. Bacteria are grown overnight(12-14 hours) at 37° C. in an atmosphere of 5% CO₂. (In this example,media was BHI agar supplemented with Leventhal's base. Other growthmedia are well known to those skilled in the art). Bacteria from ten 25mL agar plates were collected and suspended in 25 mL 10 mM Tris adjustedto ph 8.0 with HCl. An equal volume of 10 mM Tris (pH 8.0) containing 2%sarkosyl was added and the mixture mixed gently for 1 hour at 4° C. Thiswas centrifuged at 100,000×g for seventy minutes at 20° C. and thesupernatant discarded. The pellet was resuspended in 25 mL 10 mM Tris(PH 8.0) containing 1% sarkosyl by passing through a 25 gauge needle.This was centrifuged at 100,000×g for seventy minutes at 20° C. and thesupernatant discarded. The pellet was resuspended in 10 mL 10 mM Tris(pH 8.0) by passing through a 25 gauge needle. This fraction containsthe sarkosyl insoluble components of the cell, and is enriched for outermembrane proteins. (An additional step may be incorporated to removeresidual sarkosyl detergent, whereby the protein solution is dialysedfor four cycles of 4-8 hours against 100-1000 volumes of, for example,10 mM Tris.Cl pH 8.0 or PBS (phosphate buffered saline) at 4° C.

Having determined the concentration of protein in the suspension byabsorbance at wavelength of 280 nm, or by using a BCA kit (Pierce),approximately 1 mL of solution containing 10 mg of protein in a solutioncontaining 1% SDS (sodium lauryl sulphate), 2% β-mercaptoethanol wasseparated on 1.5 mm thick 6% SDS-PAGE in the BioRad mini-protean IIapparatus. The high molecular weight NhhA was eluted from the gel usingthe BioRad “mini Whole gel Eluter”. Approximately 10% of each elutedfraction was checked by SDS-PAGE separation followed by Coomassiestaining. Fractions containing NhhA essentially free of other proteinswere pooled. This procedure was carried out to isolate over expressedmature NhhA as described in Example 2 (SEQ ID NO:1), over expressedBglII deletion mature NhhA as described in Example 4 (SEQ ID NO:23) andover expressed NhhA deletion mutant as described in Example 6 (SEQ IDNO:25). Isolated protein is shown in FIG. 12.

EXAMPLE 10 Immunogenicity of Purified NhhA Deletion Mutant Polypeptides

Mice were inoculated with purified wild-type NhhA polypeptides anddeletion mutants as described in the previous Examples. In one group,each Balb/C mouse was inoculated subcutaneously with approximately 130μg PMC21 NhhA with MPL+TDM™ adjuvant (obtained from Sigma-Aldrich) onday 0, 115 μg on day 14. In a second group, each mouse was inoculatedwith approximately 120 μg protein with MPL+TDM™ adjuvant (obtained fromSigma-Aldrich) at day 0 and 190 μg at day 14. In a third group, eachmouse was inoculated with approximately 260 μg protein with MPL+TDM™adjuvant (obtained from Sigma-Aldrich) at day 0 and 1240 μg at day 14.Blood samples were taken at day 21 and serum was extracted. These serawere tested for the presence of antibodies recognising full length PMC21 NhhA by Western immunoblot (FIG. 13). OMC preparations (5 mg) of P6(overexpresses PMC21 NhhA) and Strain 2A (NhhA expression abolished)were separated by 6% SDS-PAGE using the BioRad Mini Protean IIelectrophoresis apparatus. The proteins were transferred tonitrocellulose electrophoretically, and the filter was cut into 3 mmstrips then blocked in 5% skim milk in PBS. Mouse sera was diluted to1:1000 and 1:10000 in 5% skim milk powder and incubated with thenitrocellulose strips. Antibody binding was detected usingalkaline-phosphatase conjugated anti-mouse IgG (Sigma) beforecolorimetric detection with NBT/BCIP (Sigma). As can be seen from FIG.13, it is possible to elicit an immune response against the full lengthmature PMC21 NhhA polypeptide by inoculation with NhhA deletion mutantsor with full length mature NhhA polypeptides.

EXAMPLE 11 Expression of Deletion Mutant Polypeptide in E. coli

In addition to expression of the mutant polypeptides of the invention inN. meningitidis, they may also be expressed in E. coli bacteria. Any ofthe recombinant nhhA deletion mutants of Examples 4-8 may be used astemplate for PCR amplification. Oligonucleotide primers used may be asdescribed in International Publication WO99/31132 (such as SEQ ID NO 24and SEQ ID NO 25 of that document). The amplification product may berestriction digested with BamHI/HindIII enzymes and ligated withBamHI/HindIII restriction digested plasmid pMALC2 (New England BioLabs),and the resultant plasmid transformed into competent E. coli strainDH5α. The resulting strain can be induced to express high levels ofrecombinant protein using conditions recommended by the manufacturer ofpMALC2. The resulting recombinant protein is a fusion of maltose bindingprotein and the deletion mutant NhhA polypeptide of the invention. Thismay be semi-purified by separation on SDS-PAGE followed byelectroelution using the Mini-Gel Electro-eluter (BioRad) according tomanufacturers instructions. The semi-purified fusion protein may then bedialysed against PBS, before digestion with the protease enzyme FactorXa. to cleave the maltose binding protein moiety from the recombinantNhhA protein. The recombinant NhhA protein may be purified by standardmethods, as for example described by R. K. Scopes, Protein Purification(Springer-Verlag, New York, N.Y. USA, 1993).

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. It will therefore beappreciated by those of skill in the art that, in light of the instantdisclosure, various modifications and changes can be made in theparticular embodiments exemplified without departing from the scope ofthe present invention.

All computer programs, algorithms, patent and scientific literaturereferred to herein is incorporated herein by reference.

TABLE 1 C1 V1 C2 V2 C3 V3 C4 V4 C5 Consensus 1-50 51-108 109-120 121-134135-198 199-220 221-239 240-248 249-604 SEQ ID NO: 11 PMC21 1-50 51-108109-120 121-124 125-188 189-210 211-229 230-236 237-591 SEQ ID NO: 1 H411-50 51-102 103-114 115-124 125-188 189-210 211-229 230-236 237-591 SEQID NO: 2 P20 1-50 51-105 106-117 118-121 122-185 186-205 206-224 225-234235-589 SEQ ID NO: 3 EG327 1-50 51-104 105-116 117-126 127-190 191-212213-231 232-238 239-594 SEQ ID NO: 4 EG329 1-50 51-108 109-120 121-124125-188 189-210 211-229 230-236 237-591 SEQ ID NO: 5 H38 1-50 51-105106-117 118-131 132-195 196-217 218-236 237-243 244-599 SEQ ID NO: 6 H151-50 51-104 105-116 117-130 131-194 195-216 217-235 236-242 243-598 SEQID NO: 7 BZ10 1-50 51-104 105-116 117-130 131-194 195-216 217-235236-242 243-598 SEQ ID NO: 8 BZ198 1-50 51-104 105-116 117-126 127-190191-212 213-231 232-238 239-594 SEQ ID NO: 9 Z2491 1-50 51-102 103-114115-124 125-188 189-208 209-227 228-236 237-592 SEQ ID NO: 10

TABLE 2 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile, Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

1. An isolated protein comprising: (A) one or more conserved regions ofan NhhA polypeptide which one or more conserved regions respectivelyconsist of any of the following amino acid sequences selected from thegroup consisting of: (i) residues 1 to 50 of SEQ ID NO:11; (ii) residues109 to120 of SEQ ID NO:11; (iii) residues 135 to198 of SEQ ID NO:11;(iv) residues 221 to 239 of SEQ ID NO:11; and (v) residues 249 to 604 ofSEQ ID NO:11; and (B) one or more variable regions of an NhhApolypeptide, the one or more variable regions respectively consisting ofany of the following amino acid sequences selected from the groupconsisting of: (a) residues 121 to134 of SEQ ID NO:11; (b) residues 199to 220 of SEQ ID NO:11; and (c) residues 240 to 248 of SEQ ID NO:11;wherein a variable region of residues 51-108 of SEQ ID NO:11 or afragment consisting of at least six amino acids thereof is absent;wherein the isolated protein is capable of eliciting an immune responseagainst a plurality of strains of Neisseria meningitidis.
 2. An isolatedprotein consisting of two or more contiguous conserved regions of anNhhA polypeptide, the two or more conserved regions respectivelyconsisting of-an any of the following amino acid sequences selected fromthe group consisting of: (i) residues 1 to 50 of SEQ ID NO:11; (ii)residues 109 to120 of SEQ ID NO:11; (iii) residues 135 to198 of SEQ IDNO:11; (iv) residues 221 to 239 of SEQ ID NO:11; and (v) residues 249 to604 of SEQ ID NO:11; wherein the isolated protein is capable ofeliciting an immune response against a plurality of strains of Neisseriameningitidis.
 3. The isolated protein of claim 1, wherein the protein isa deletion mutant of the NhhA polypeptide.
 4. The isolated protein ofclaim 2, wherein the protein is a deletion mutant of the NhhApolypeptide.
 5. The isolated protein of claim 1, wherein the conservedregion amino acid sequence is selected from the group consisting of: (I)residues 1 to 50 of SEQ ID NO:1; (II) residues 1 to 50 of SEQ ID NO:2;(III) residues 1 to 50 of SEQ ID NO:3; (IV) residues 1 to 50 of SEQ IDNO:4; (V) residues 1 to 50 of SEQ ID NO:5; (VI) residues 1 to 50 of SEQID NO:6; (VII) residues 1 to 50 of SEQ ID NO:7; (VIII) residues 1 to 50of SEQ ID NO:8; (IX) residues 1 to 50 of SEQ ID NO:9; (X) residues 1 to50 of SEQ ID NO:10; (XI) residues 109 to 120 of SEQ ID NO:1; (XII)residues 103 to 114 of SEQ ID NO:2; (XIII) residues 106 to 117 of SEQ IDNO:3; (XIV) residues 105 to 116 of SEQ ID NO:4; (XV) residues 109 to 120of SEQ ID NO:5; (XVI) residues 106 to 117 of SEQ ID NO:6; (XVII)residues 105 to 116 of SEQ ID NO:7; (XVIII) residues 105 to 116 of SEQID NO:8; (XIX) residues 105 to 116 of SEQ ID NO:9; (XX) residues 103 to114 of SEQ ID NO:10; (XXI) residues 125 to 188 of SEQ ID NO:1; (XXII)residues 125 to 188 of SEQ ID NO:2; (XXIII) residues 122 to 185 of SEQID NO:3; (XXIV) residues 127 to 190 of SEQ ID NO:4; (XXV) residues 125to 188 of SEQ ID NO:5; (XXVI) residues 132 to 195 of SEQ ID NO:6;(XXVII) residues 131 to 194 of SEQ ID NO:7; (XXVIII) residues 131 to 194of SEQ ID NO:8; (XXIX) residues 127 to 190 of SEQ ID NO:9; (XXX)residues 125 to 188 of SEQ ID NO:10; (XXXI) residues 211 to 229 of SEQID NO:1; (XXXII) residues 211 to 229 of SEQ ID NO:2; (XXXIII) residues206 to 224 of SEQ ID NO:3; (XXXIV) residues 213 to 231 of SEQ ID NO:4;(XXXV) residues 211 to 229 of SEQ ID NO:5; (XXXVI) residues 218 to 236of SEQ ID NO:6; (XXXVII) residues 217 to 235 of SEQ ID NO:7; (XXXVIII)residues 217 to 235 of SEQ ID NO:8; (XXXIX) residues 213 to 231 of SEQID NO:9; (XL) residues 209 to 227 of SEQ ID NO:10; (XLI) residues 237 to591 of SEQ ID NO:1; (XLII) residues 237 to 592 of SEQ ID NO:2; (XLIII)residues 235 to 589 of SEQ ID NO:3; (XLIV) residues 239 to 594 of SEQ IDNO:4; (XLV) residues 237 to 591 of SEQ ID NO:5; (XLVI) residues 244 to599 of SEQ ID NO:6; (XLVII) residues 243 to 598 of SEQ ID NO:7; (XLVIII)residues 243 to 598 of SEQ ID NO:8. (XLIX) residues 239 to 594 of SEQ IDNO:9; and (L) residues 237 to 592 of SEQ ID NO:10.
 6. The isolatedprotein of claim 2, wherein the conserved region amino acid sequence isselected from the group consisting of: (I) residues 1 to 50 of SEQ IDNO:1; (II) residues 1 to 50 of SEQ ID NO:2; (III) residues 1 to 50 ofSEQ ID NO:3; (IV) residues 1 to 50 of SEQ ID NO:4; (V) residues 1 to 50of SEQ ID NO:5; (VI) residues 1 to 50 of SEQ ID NO:6; (VII) residues 1to 50 of SEQ ID NO:7; (VIII) residues 1 to 50 of SEQ ID NO:8; (IX)residues 1 to 50 of SEQ ID NO:9; (X) residues 1 to 50 of SEQ ID NO:10;(XI) residues 109 to 120 of SEQ ID NO:1; (XII) residues 103 to 114 ofSEQ ID NO:2; (XIII) residues 106 to 117 of SEQ ID NO:3; (XIV) residues105 to 116 of SEQ ID NO:4; (XV) residues 109 to 120 of SEQ ID NO:5;(XVI) residues 106 to 117 of SEQ ID NO:6; (XVII) residues 105 to 116 ofSEQ ID NO:7; (XVIII) residues 105 to 116 of SEQ ID NO:8; (XIX) residues105 to 116 of SEQ ID NO:9; (XX) residues 103 to 114 of SEQ ID NO:10;(XXI) residues 125 to 188 of SEQ ID NO:1; (XXII) residues 125 to 188 ofSEQ ID NO:2; (XXII) residues 122 to 185 of SEQ ID NO:3; (XXIV) residues127 to 190 of SEQ ID NO:4; (XXV) residues 125 to 188 of SEQ ID NO:5;(XXVI) residues 132 to 195 of SEQ ID NO:6; (XXVII) residues 131 to 194of SEQ ID NO:7; (XXVIII) residues 131 to 194 of SEQ ID NO:8; (XXIX)residues 127 to 190 of SEQ ID NO:9; (XXX) residues 125 to 188 of SEQ IDNO:10; (XXXI) residues 211 to 229 of SEQ ID NO:1; (XXXII) residues 211to 229 of SEQ ID NO:2; (XXXIII) residues 206 to 224 of SEQ ID NO:3;(XXXIV) residues 213 to 231 of SEQ ID NO:4; (XXXV) residues 211 to 229of SEQ ID NO:5; (XXXVI) residues 218 to 236 of SEQ ID NO:6; (XXXVII)residues 217 to 235 of SEQ ID NO:7; (XXXVIII) residues 217 to 235 of SEQID NO:8; (XXXIX) residues 213 to 231 of SEQ ID NO:9; (XL) residues 209to 227 of SEQ ID NO:10; (XLI) residues 237 to 591 of SEQ ID NO:1; (XLII)residues 237 to 592 of SEQ ID NO:2; (XLIII) residues 235 to 589 of SEQID NO:3; (XLIV) residues 239 to 594 of SEQ ID NO:4; (XLV) residues 237to 591 of SEQ ID NO:5; (XLVI) residues 244 to 599 of SEQ ID NO:6;(XLVII) residues 243 to 598 of SEQ ID NO:7; (XLVIII) residues 243 to 598of SEQ ID NO:8. (XLIX) residues 239 to 594 of SEQ ID NO:9; and (L)residues 237 to 592 of SEQ ID NO:10.
 7. The isolated protein of claim 1,wherein the variable region amino acid sequence is selected from thegroup consisting of: (AA) residues 121 to 124 of SEQ ID NO:1; (AB)residues 115 to 124 of SEQ ID NO:2; (AC) residues 118 to 121 of SEQ IDNO:3; (AD) residues 117 to 126 of SEQ ID NO:4; (AE) residues 121 to 124of SEQ ID NO:5; (AF) residues 118 to 131 of SEQ ID NO:6; (AG) residues117 to 130 of SEQ ID NO:7; (AH) residues 117 to 130 of SEQ ID NO:8; (AI)residues 117 to 126 of SEQ ID NO:9; (AJ) residues 115 to 124 of SEQ IDNO:10; (BA) residues 189 to 210 of SEQ ID NO:1; (BB) residues 189 to 210of SEQ ID NO:2; (BC) residues 186 to 205 of SEQ ID NO:3; (BD) residues191 to 212 of SEQ ID NO:4; (BE) residues 189 to 210 of SEQ ID NO:5; (BF)residues 196 to 217 of SEQ ID NO:6; (BG) residues 195 to 216 of SEQ IDNO:7; (BH) residues 195 to 216 of SEQ ID NO:8; (BI) residues 191 to 212of SEQ ID NO:9; (BJ) residues 189 to 208 of SEQ ID NO:10; (CA) residues230 to 236 of SEQ ID NO:1; (CB) residues 230 to 236 of SEQ ID NO:2; (CC)residues 225 to 234 of SEQ ID NO:3; (CD) residues 232 to 238 of SEQ IDNO:4; (CE) residues 230 to 236 of SEQ ID NO:5; (CF) residues 237 to 243of SEQ ID NO:6; (CG) residues 236 to 242 of SEQ ID NO:7; (CH) residues236 to 242 of SEQ ID NO:8; (CI) residues 232 to 238 of SEQ ID NO:9; and(CJ) residues 228 to 236 of SEQ ID NO:10.
 8. The isolated protein ofclaim 1, comprising any of the following amino acid sequences selectedfrom the group consisting of: SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:33;SEQ ID NO:36 and SEQ ID NO:38.
 9. The isolated protein of claim 2,comprising any of the following amino acid sequences selected from thegroup consisting of SEQ ID NO:25; SEQ ID NO:27 and SEQ ID NO:39.
 10. Apharmaceutical composition comprising one or more isolated proteinsaccording to claim 1 and a pharmaceutically acceptable carrier, diluentor excipient.
 11. A pharmaceutical composition comprising one or moreisolated proteins according to claim 2 and a pharmaceutically acceptablecarrier, diluent or excipient.
 12. A method of therapeutically treatingan N. meningitidis infection in a mammal including administering apharmaceutically effective amount of the pharmaceutical composition ofclaim 10 to the mammal to thereby therapeutically treat said N.meningitidis infection.
 13. A method of therapeutically treating anNeisseria meningitidis infection in a mammal including administering apharmaceutically effective amount of the pharmaceutical composition ofclaim 11 to the mammal to thereby therapeutically treat the Neisseriameningitidis infection.
 14. The method of claim 12, whereinadministering the pharmaceutically effective amount of thepharmaceutical composition elicits an immune response to Neisseriameningitidis.
 15. The method of claim 12, wherein the mammal is a human.16. The method of claim 13, wherein the mammal is a human.